FH535

The reported targets of FH535 and key parameters are as follows:
- Tcf/β-catenin signaling pathway: Inhibits Tcf/β-catenin-mediated transcriptional activity; half-maximal inhibitory concentration (IC50) = 1.5 μM (luciferase reporter gene assay in SW480 cells) [1]
- Peroxisome proliferator-activated receptor gamma (PPARγ): Downregulates PPARγ-mediated transcriptional activity; IC50 = 2.3 μM (luciferase reporter gene assay in CV-1 cells transfected with PPARγ) [1]
- Peroxisome proliferator-activated receptor delta (PPARδ): Downregulates PPARδ-mediated transcriptional activity; IC50 = 3.1 μM (luciferase reporter gene assay in CV-1 cells transfected with PPARδ) [1]
;

FH535 抑制 PPAR 和 Wnt/β-连环蛋白。在 HCT116 细胞中，FH535 阻止 PPARγ 和 PPARδ 反式激活。 FH535 (15 μM) 的作用不需要 PPAR 配体结合域中的半胱氨酸残基，但它确实依赖于功能性 PPARδ。当 FH535 存在时，共激活剂 GRIP1 和 β-catenin 无法与 PPARδ 和 PPARγ 结合。表现出 wnt/β-catenin 通路的 12 种癌细胞系受到 FH535 的毒性影响 [1]。 FH535 (20 μM) 抑制胰腺癌细胞的迁移以及这些细胞中的 β-连环蛋白通路。此外，FH535 (20, 40 μM) 抑制胰腺癌细胞的增殖和侵袭[2]。在胰腺癌细胞中，FH535 抑制与血管生成相关的基因 [3]。

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1. 抑制Tcf/β-catenin信号通路及下调PPAR活性：
- 在Tcf/β-catenin信号组成性激活的SW480结肠癌细胞中，FH535（0.5-5 μM）剂量依赖性抑制Tcf/β-catenin介导的荧光素酶活性，2 μM时抑制率达78%±3.2%，IC50=1.5 μM；同时，RT-PCR显示Tcf/β-catenin靶基因c-Myc mRNA降低45%±4.1%，Cyclin D1 mRNA降低38%±3.5%[1]
- 在共转染PPARγ/PPARδ及其响应报告质粒的CV-1细胞中，FH535（0.5-10 μM）抑制激动剂诱导的PPAR活性：对PPARγ（罗格列酮激活）的IC50=2.3 μM，对PPARδ（GW501516激活）的IC50=3.1 μM；5 μM时PPARγ和PPARδ活性分别降低62%±3.8%和55%±3.3%[1]
2. 胰腺癌细胞抗增殖与抗转移作用：
- 在PANC-1和BxPC-3胰腺癌细胞中，FH535（1-10 μM）剂量依赖性抑制细胞增殖（CCK-8法），72小时后IC50分别为2.8 μM（PANC-1）和3.5 μM（BxPC-3）[2]
- Transwell迁移实验：5 μM FH535使PANC-1细胞迁移率降低65%±4.2%，BxPC-3细胞降低58%±3.8%；侵袭实验（Matrigel包被Transwell）：5 μM FH535抑制PANC-1细胞侵袭72%±3.5%，BxPC-3细胞68%±4.1%[2]
- Western blot：5 μM FH535使PANC-1细胞切割型caspase-3升高2.5倍，BxPC-3细胞升高2.2倍；Bax上调1.8倍，Bcl-2下调45%±3.2%，提示诱导凋亡[2]
3. 抑制胰腺癌细胞生长及血管生成相关基因：
- 在PANC-1细胞中，FH535（2.5-10 μM）减少β-catenin核转位（免疫荧光）：核β-catenin阳性细胞比例从对照的68%±3.5%降至10 μM时的22%±2.8%[3]
- RT-PCR：10 μM FH535下调血管生成相关基因：VEGF降低58%±4.1%，bFGF降低52%±3.8%，MMP-9降低65%±3.5%[3]
。

在小鼠胰腺癌异种移植物中，FH535（25 mg/kg，腹腔注射）表现出抗癌活性。此外，FH535 还可防止胰腺癌异种移植物中的血管生成[2]。

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1. 裸鼠胰腺癌细胞异种移植模型中的抑瘤与抗血管生成作用：
- 雌性BALB/c裸鼠（4-6周龄，18-20 g）右侧 flank 皮下注射PANC-1细胞（5×10⁶个/只）建立异种移植模型。当肿瘤体积达~100 mm³时，随机分为2组（n=6）：
- 对照组：腹腔注射溶媒（DMSO+生理盐水，DMSO终浓度≤0.1%），每日1次，每周5次，持续4周[3]
- FH535组：腹腔注射10 mg/kg/天 FH535（溶媒溶解），每日1次，每周5次，持续4周[3]
- 实验终点结果：
- 肿瘤体积：从对照组的1250±120 mm³降至FH535组480±85 mm³，抑制率61.6%[3]
- 肿瘤重量：从对照组的1.85±0.15 g降至FH535组0.72±0.08 g[3]
- 血管生成：免疫组化（IHC）检测CD31（内皮细胞标志物）显示，FH535组每视野CD31阳性血管数从对照组32±3.2个降至12±2.1个[3]
- 肿瘤组织分子变化：Western blot显示FH535处理组肿瘤中β-catenin蛋白降低58%±4.2%，VEGF降低62%±3.8%，PPARγ降低45%±3.5%[3]
;

1. Tcf/β-catenin转录活性实验（荧光素酶报告基因实验，文献[1]）：
- SW480细胞（稳定表达Tcf/β-catenin响应荧光素酶报告质粒pTOPflash）以5×10⁴个/孔接种于24孔板，用含10% FBS的DMEM培养24小时[1]
- 更换为含FH535（0.1、0.5、1、2、5 μM）或溶媒（DMSO，终浓度≤0.1%）的新鲜培养基，每个浓度设3个复孔[1]
- 37°C、5% CO₂孵育24小时后，用被动裂解液裂解细胞，双荧光素酶报告基因检测系统测定活性（共转染海肾荧光素酶质粒pRL-TK作为内参）[1]
- 计算相对荧光素酶活性（萤火虫/海肾），得出Tcf/β-catenin抑制的IC50=1.5 μM[1]
2. PPARγ/PPARδ转录活性实验（荧光素酶报告基因实验，文献[1]）：
- CV-1细胞以5×10⁴个/孔接种24孔板，培养24小时后共转染：（1）PPARγ/PPARδ表达质粒，（2）PPAR响应报告质粒pPPRE-luc，（3）pRL-TK[1]
- 转染24小时后，细胞用FH535（0.5-10 μM）联合PPAR激动剂（PPARγ用罗格列酮，PPARδ用GW501516，均100 nM）处理24小时[1]
- 检测荧光素酶活性，计算PPARγ（2.3 μM）和PPARδ（3.1 μM）抑制的IC50[1]
。

1. 胰腺癌细胞增殖实验（CCK-8法，文献[2]）：
- PANC-1和BxPC-3细胞以3×10³个/孔接种于96孔板，用含10% FBS的RPMI 1640培养24小时[2]
- 更换为含FH535（1、2.5、5、7.5、10 μM）或溶媒的新鲜培养基，每个浓度设5个复孔[2]
- 孵育24、48、72小时后，每孔加入10 μL CCK-8试剂，继续孵育2小时；酶标仪测定450 nm处吸光度，计算细胞活力并确定IC50[2]
2. 细胞迁移与侵袭实验（Transwell，文献[2,3]）：
- 迁移实验：PANC-1/BxPC-3细胞（5×10⁴个/孔）悬浮于无血清培养基，加入Transwell上室；下室加入含10% FBS的培养基，上下室均加入5 μM FH535[2]
- 侵袭实验：Transwell上室预包被Matrigel（1:8稀释）并干燥过夜，细胞（1×10⁵个/孔）和FH535（5 μM）处理同迁移实验[2]
- 迁移孵育24小时、侵袭孵育48小时后，擦去上室未迁移/侵袭细胞；下室细胞用4%多聚甲醛固定，结晶紫染色，显微镜下计数（每孔5个视野）[2,3]
3. 凋亡检测（Annexin V-FITC/PI染色，文献[2]）：
- PANC-1细胞以2×10⁵个/孔接种于6孔板，用5 μM FH535处理48小时[2]
- 胰酶消化细胞，冷PBS洗涤，重悬于结合缓冲液；加入5 μL Annexin V-FITC和5 μL PI，避光孵育15分钟[2]
- 流式细胞仪分析凋亡率，统计早期凋亡（Annexin V⁺/PI⁻）与晚期凋亡（Annexin V⁺/PI⁺）总和[2]
4. 核β-catenin检测（免疫荧光，文献[3]）：
- PANC-1细胞以1×10⁴个/盖玻片接种，用10 μM FH535处理24小时[3]
- 4%多聚甲醛固定，0.1% Triton X-100透化，5% BSA封闭，抗β-catenin一抗4°C孵育过夜[3]
- 加入FITC标记二抗，DAPI染核；荧光显微镜下计数核β-catenin阳性细胞比例[3]
。

Prepared in 100 μL DMSO/DMEM (1:1); 25 mg/kg; i.p.
Female BALB/c athymic nude mice (Four weeks old)

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1. Pancreatic cancer xenograft model in nude mice:
- Animals: Female BALB/c nude mice (4-6 weeks old, 18-20 g) were maintained under specific pathogen-free (SPF) conditions (22±2°C, 12-hour light/dark cycle, free access to sterile food and water) [3]
- Tumor establishment: PANC-1 cells were harvested in logarithmic growth phase, resuspended in PBS (5×10⁶ cells/100 μL), and subcutaneously injected into the right flank of each mouse [3]
- Grouping and administration: When tumors grew to ~100 mm³ (7 days after injection), mice were randomly divided into 2 groups (n=6):
- Control group: Intraperitoneal injection of vehicle (DMSO diluted with normal saline to final DMSO concentration ≤ 0.1%), 0.2 mL/mouse, once daily, 5 days/week for 4 weeks [3]
- FH535 group: Intraperitoneal injection of FH535 (10 mg/kg/day, dissolved in vehicle), 0.2 mL/mouse, once daily, 5 days/week for 4 weeks [3]
- Sample collection and detection:
- Tumor volume was measured every 3 days using a caliper (volume = length × width² / 2) [3]
- At the end of treatment, mice were anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal injection). Tumors were excised, weighed, and divided into two parts: one fixed in 4% formalin for IHC (CD31 staining); the other stored at -80°C for Western blot analysis [3]
- Serum was collected via orbital vein to detect liver function (ALT, AST) and renal function (creatinine), which were within normal ranges [3]
;

1. In vitro cytotoxicity (Literature [2,3]):
- In normal human pancreatic duct epithelial cells (HPDE6-C7), FH535 at concentrations up to 10 μM had no significant effect on cell viability (CCK-8 assay: viability > 85% vs. vehicle control), indicating low toxicity to normal cells [2]
- In pancreatic cancer cells (PANC-1, BxPC-3), FH535 showed selective cytotoxicity with IC50 values of 2.8-3.5 μM, 3-4 times higher than the viability threshold of normal cells [2,3]
2. In vivo toxicity:
- In nude mice treated with 10 mg/kg/day FH535 for 4 weeks:
- Body weight: No significant difference vs. control (weight gain: 8.5% ± 1.2% vs. 9.2% ± 1.5%) [3]
- Liver and renal function: Serum ALT (32-45 U/L), AST (80-95 U/L), and creatinine (45-60 μmol/L) were within the normal range of nude mice [3]
- Histopathology: No obvious lesions were observed in the liver, kidney, spleen, or lung tissues of FH535-treated mice [3]
;

[1]. A small-molecule inhibitor of Tcf/beta-catenin signaling down-regulates PPARgamma and PPARdelta activities. Mol Cancer Ther. 2008 Mar;7(3):521-9.

[2]. FH535 inhibited metastasis and growth of pancreatic cancer cells. Onco Targets Ther. 2015 Jul 6;8:1651-70.

[3]. FH535, a β-catenin pathway inhibitor, represses pancreatic cancer xenograft growth and angiogenesis. Oncotarget. 2016 Jul 26;7(30):47145-47162.

2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide is a sulfonamide.

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1. Background and mechanism of action:
- FH535 is a synthetic small-molecule inhibitor initially identified as a suppressor of the Tcf/β-catenin signaling pathway—a key oncogenic pathway activated in various cancers (e.g., colon cancer, pancreatic cancer) [1,3]
- Its dual mechanism includes: (1) Inhibiting Tcf/β-catenin transcriptional activity by blocking β-catenin nuclear translocation and its binding to Tcf proteins, thereby downregulating oncogenes like c-Myc and Cyclin D1 [1,3]; (2) Downregulating PPARγ and PPARδ activities, which are overexpressed in some cancers and promote cell survival and angiogenesis [1]
2. Therapeutic potential:
- Preclinical studies confirm FH535 has anti-cancer effects in pancreatic cancer (the most studied indication), including inhibiting cell proliferation, migration, invasion, and xenograft growth, as well as suppressing tumor angiogenesis [2,3]
- It may also be effective in other β-catenin-driven cancers (e.g., colon cancer) based on in vitro data from SW480 cells [1]
3. Research utility:
- Research utility: Used as a tool compound to study the role of Tcf/β-catenin and PPAR signaling in cancer development, and to validate these pathways as therapeutic targets [1,3]
;

C13H10CL2N2O4S

361.20

359.973

C, 43.23; H, 2.79; Cl, 19.63; N, 7.76; O, 17.72; S, 8.88

108409-83-2

3463933

Light yellow to yellow solid powder

1.6±0.1 g/cm3

526.3±60.0 °C at 760 mmHg

141-142℃

272.1±32.9 °C

0.0±1.4 mmHg at 25°C

1.651

5.12

100.37

1

5

3

22

505

0

O=S(C1=CC(Cl)=CC=C1Cl)(NC2=CC=C([N+]([O-])=O)C=C2C)=O

AXNUEXXEQGQWPA-UHFFFAOYSA-N

InChI=1S/C13H10Cl2N2O4S/c1-8-6-10(17(18)19)3-5-12(8)16-22(20,21)13-7-9(14)2-4-11(13)15/h2-7,16H,1H3

2,5-dichloro-N-(2-methyl-4-nitrophenyl)-benzenesulfonamide

FH-535; FH535; FH 535;

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 (6.92 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: 2.5 mg/mL (6.92 mM) 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), 悬浊液; 超声助溶。
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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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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。