KNK437

In human colon carcinoma cells, KNK437 acts as an inhibitor of the induction of heat shock proteins (HSPs), including HSP70 and HSP90 [1]
- In murine transplantable tumor models, KNK437 inhibits the synthesis of heat shock proteins (HSPs) to suppress the acquisition of thermotolerance [2]
- In human oral squamous cell carcinoma cells, KNK437 inhibits heat-induced induction of heat shock proteins (HSPs) and modulates heat-induced histone H3 methylation [3]

在 COLO 320DM（人结肠癌）细胞中，KNK437 抑制多种 HSP 的激活，例如 HSP105、HSP70 和 HSP40。初始热处理后，COLO 320DM 细胞的耐热能力被 KNK437 (100 μM) 抑制。在 COLO 320DM 细胞 (0-200 μM) 和 HeLa S3 细胞 (100, 200 μM) 中，KNK437 表现出剂量依赖性的耐热性抑制 [1]。在加热前和加热后应用时，KNK437 (100 μM) 抑制 HSC4 和 KB 细胞中 H3-Lys4 的甲基化，但对 H3 Lys9 甲基化没有影响。 HSP70 的表达也受到 KNK437 的抑制 [3]。

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人结肠癌细胞实验（HT-29细胞系）：将HT-29细胞用10 μM、50 μM、100 μM的KNK437预处理1小时后，在43°C下热休克30分钟，KNK437 可剂量依赖性地抑制HSP70和HSP90的诱导表达（通过Western blot检测）。此外，KNK437 还降低了HT-29细胞热耐受性的获得：100 μM KNK437 联合热休克处理组的细胞存活率为23.5%（热休克单独处理组为68.2%，通过MTT法检测） [1]
- 人口腔鳞癌细胞实验（HSC-2细胞系）：HSC-2细胞用50 μM KNK437 处理1小时后，在43°C下热休克45分钟。KNK437 显著抑制热诱导的组蛋白H3二甲基化（Lys9）和三甲基化（Lys9）水平（通过Western blot检测），同时抑制热诱导的HSP70和HSP27表达，并降低热休克后的细胞存活能力（KNK437 联合热休克组的克隆形成存活率为18.7%，热休克单独组为49.3%） [3]

它是一种弱剂，KNK437。给予 KNK437（62.5-400 mg/kg）后，无肿瘤 CD-1 (ICR) 小鼠体重恢复。在不耐热的肿瘤中，KNK437 (200 mg/kg) 不会增强热敏感性或具有任何抗癌作用。 KNK437 可协同增强 44°C 200 mg/kg 分级热处理的抗癌作用。在开始加热过程前六小时施用 KNK437（200 mg/kg，腹膜内注射）会降低耐热性 [2]。

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小鼠可移植肿瘤模型（BALB/c小鼠结肠26腺癌）：将皮下接种结肠26肿瘤（体积约100 mm³）的小鼠分为4组：对照组、热休克单独组、KNK437 单独组、KNK437 联合热休克组。在肿瘤局部热休克（43°C，45分钟）前1小时，通过腹腔注射给予KNK437（100 mg/kg）。KNK437 联合热休克组的肿瘤生长受到显著抑制：处理后14天的肿瘤体积为285 mm³（热休克单独组为642 mm³）。肿瘤组织的免疫组化染色显示，KNK437 降低了热诱导的肿瘤细胞HSP70表达；KNK437 单独组未观察到明显的肿瘤退缩 [2]

HT-29细胞培养及热耐受性检测：HT-29细胞在含10%胎牛血清、100 U/mL青霉素和100 μg/mL链霉素的RPMI 1640培养基中，于37°C、5% CO₂培养箱中培养。为诱导热耐受性，将细胞接种于96孔板（5×10³个细胞/孔），培养24小时后，用KNK437（0 μM、10 μM、50 μM、100 μM）处理1小时，随后在水浴中43°C热休克30分钟。热休克后，细胞在37°C培养48小时，通过MTT法检测细胞存活：每孔加入20 μL MTT溶液（5 mg/mL），孵育4小时后，加入150 μL DMSO溶解甲瓒结晶，在570 nm处测定吸光度 [1]
- HT-29细胞中HSP的Western blot检测：KNK437 和热休克处理后，用冷PBS洗涤HT-29细胞，并用含蛋白酶抑制剂的RIPA缓冲液裂解细胞。细胞裂解液在4°C、12,000×g条件下离心15分钟，收集上清液；通过BCA法测定蛋白浓度，取30 μg蛋白用10% SDS-PAGE分离，转移至PVDF膜。膜用含5%脱脂牛奶的TBST封闭1小时（室温），随后用抗HSP70、HSP90和β-肌动蛋白（内参）的一抗4°C孵育过夜；TBST洗涤后，用辣根过氧化物酶标记的二抗室温孵育1小时，通过增强化学发光（ECL）试剂盒显影，并用密度分析法定量条带 [1]
- HSC-2细胞克隆形成实验：将HSC-2细胞接种于6孔板（2×10³个细胞/孔），培养24小时后，用50 μM KNK437 处理1小时，随后43°C热休克45分钟。热休克后，细胞在37°C培养10天以形成克隆；用4%多聚甲醛固定15分钟，0.1%结晶紫染色30分钟，计数含50个以上细胞的克隆，克隆形成存活率按（处理组克隆数/对照组克隆数）×100%计算 [3]
- HSC-2细胞中组蛋白H3甲基化的Western blot检测：HSC-2细胞按上述方法经KNK437 和热休克处理后，制备细胞裂解液，取等量蛋白用12% SDS-PAGE分离；膜用抗二甲基组蛋白H3（Lys9）、三甲基组蛋白H3（Lys9）和β-肌动蛋白的一抗孵育，随后加入二抗并通过ECL显影，分析条带强度以定量组蛋白甲基化水平 [3]

Dissolved in Olive oil; 200 mg/kg; i.p. injection
C3H/He mice

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Murine tumor model establishment and treatment: Female BALB/c mice (6-8 weeks old) were used. Colon 26 adenocarcinoma cells (5×10⁶ cells in 0.1 mL PBS) were injected subcutaneously into the right flank of each mouse. Tumor volume was measured every 2 days using a caliper, calculated as (length × width²) / 2. When tumors reached a volume of ~100 mm³, mice were randomly assigned to four groups (n=6 per group): 1) Control group: intraperitoneal injection of vehicle (0.1 mL DMSO diluted 1:10 in PBS); 2) Heat shock alone group: vehicle injection 1 hour before local tumor heat shock; 3) KNK437 alone group: intraperitoneal injection of KNK437 (100 mg/kg, dissolved in DMSO and diluted with PBS to a final DMSO concentration of 10%); 4) KNK437 + heat shock group: KNK437 injection (100 mg/kg) 1 hour before heat shock. Local tumor heat shock was performed by immersing the tumor-bearing flank in a 43°C water bath for 45 minutes. Tumor volume and mouse body weight were monitored every 2 days for 14 days. On day 14, mice were euthanized, and tumor tissues were excised for immunohistochemical analysis [2]
- Immunohistochemistry of murine tumor tissues: Excised tumor tissues were fixed in 10% neutral buffered formalin for 24 hours, embedded in paraffin, and cut into 4-μm sections. Sections were deparaffinized with xylene and rehydrated through graded ethanol. Antigen retrieval was performed by boiling sections in citrate buffer (pH 6.0) for 15 minutes. Sections were blocked with 3% H₂O₂ for 10 minutes to inhibit endogenous peroxidase activity, then incubated with primary antibody against HSP70 overnight at 4°C. After washing, sections were incubated with biotinylated secondary antibody for 30 minutes, followed by streptavidin-horseradish peroxidase complex for 30 minutes. Staining was visualized with 3,3'-diaminobenzidine (DAB) solution, and sections were counterstained with hematoxylin. HSP70-positive cells were counted in five random high-power fields (×400) per section [2]

In the murine Colon 26 tumor model, intraperitoneal administration of KNK437 at 100 mg/kg (once) did not cause obvious acute toxicity: no mouse death was observed, and the body weight of mice in the KNK437 alone group was comparable to that of the control group (no significant weight loss) during the 14-day observation period [2]

[1]. Benzylidene lactam compound, KNK437, a novel inhibitor of acquisition of thermotolerance and heat shock protein induction in human colon carcinoma cells. Cancer Res. 2000 Jun 1;60(11):2942-8.

[2]. The effects of KNK437, a novel inhibitor of heat shock protein synthesis, on the acquisition of thermotolerance in a murine transplantable tumor in vivo. Clin Cancer Res. 2001 Jan;7(1):215-9.

[3]. Effects of KNK437 on heat-induced methylation of histone H3 in human oral squamous cell carcinoma cells. Int J Hyperthermia. 2006 Dec;22(8):729-35.

KNK437 is a novel benzylidene lactam compound that was first identified as an inhibitor of thermotolerance acquisition and HSP induction in human colon carcinoma cells, suggesting its potential as a sensitizer for heat-based cancer therapies [1]
- The inhibitory effect of KNK437 on HSP synthesis and thermotolerance was confirmed in an in vivo murine transplantable tumor model, supporting its translational potential for improving the efficacy of clinical tumor heat therapy [2]
- In human oral squamous cell carcinoma cells, KNK437 not only inhibits HSP induction but also modulates heat-induced histone H3 methylation, indicating a possible role in regulating chromatin remodeling associated with heat stress response in cancer cells [3]

C13H11NO4

245.23

245.068

218924-25-5

9859662

Light yellow to yellow solid powder

1.5±0.1 g/cm3

412.4±55.0 °C at 760 mmHg

203.2±31.5 °C

0.0±1.0 mmHg at 25°C

1.744

1.57

55.84

0

4

1

18

393

0

C\1CN(C(=O)/C1=C\C2=CC3=C(C=C2)OCO3)C=O

NJBBLOIWMSYVCQ-VZTVMPNDSA-N

InChI=1S/C33H41N5O6S2/c1-33(2,3)37-31(42)26-19-46-20-38(26)32(43)29(40)24(15-21-9-6-5-7-10-21)36-30(41)25(18-45-4)35-28(39)17-44-27-12-8-11-22-16-34-14-13-23(22)27/h5-14,16,24-26,29,40H,15,17-20H2,1-4H3,(H,35,39)(H,36,41)(H,37,42)/t24-,25-,26-,29-/m0/s1

(Z)-3-(benzo[d][1,3]dioxol-5-ylmethylene)-2-oxopyrrolidine-1-carbaldehyde

KNK437; KNK 437; KNK-437;

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: 1.67 mg/mL (6.81 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 16.7 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: Olive oil: 30 mg/mL

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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。