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]

In COLO 320DM (human colon cancer) cells, KNK437 suppresses the activation of multiple HSPs, such as HSP105, HSP70, and HSP40. Following the initial heat treatment, COLO 320DM cells' ability to tolerate heat is inhibited by KNK437 (100 μM). In COLO 320DM cells (0-200 μM) and HeLa S3 cells (100, 200 μM), KNK437 exhibits dose-dependent inhibition of thermotolerance [1]. When applied pre- and post-heat, KNK437 (100 μM) inhibits the methylation of H3-Lys4 in HSC4 and KB cells, but has no effect on H3 Lys9 methylation. HSP70 expression is also inhibited by KNK437 [3].

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Human colon carcinoma cell experiments (HT-29 cell line): When HT-29 cells were pretreated with KNK437 at concentrations of 10 μM, 50 μM, and 100 μM for 1 hour followed by heat shock at 43°C for 30 minutes, KNK437 dose-dependently inhibited the induction of HSP70 and HSP90 (detected by Western blot). Additionally, KNK437 reduced the acquisition of thermotolerance in HT-29 cells: the survival rate of cells treated with 100 μM KNK437 plus heat shock was 23.5% (vs. 68.2% in heat shock alone group, measured by MTT assay) [1]
- Human oral squamous cell carcinoma cell experiments (HSC-2 cell line): HSC-2 cells were treated with KNK437 (50 μM) for 1 hour prior to heat shock at 43°C for 45 minutes. KNK437 significantly suppressed heat-induced increases in dimethylated histone H3 (Lys9) and trimethylated histone H3 (Lys9) (detected by Western blot). It also inhibited heat-induced induction of HSP70 and HSP27, and reduced cell survival after heat shock (the clonogenic survival rate was 18.7% in KNK437 + heat shock group vs. 49.3% in heat shock alone group) [3]

It is a weak agent, KNK437. Tumor-free CD-1 (ICR) mice regain body weight loss when given KNK437 (62.5-400 mg/kg). In tumors that were heat intolerant, KNK437 (200 mg/kg) did not enhance heat sensitivity or have any anticancer effects. The anticancer effect of fractionated heat treatment at 200 mg/kg at 44°C is synergistically enhanced by KNK437. The administration of KNK437 (200 mg/kg, i.p.) six hours prior to the beginning heating process reduces thermotolerance [2].

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Murine transplantable tumor model (Colon 26 adenocarcinoma in BALB/c mice): Mice bearing subcutaneous Colon 26 tumors (volume ~100 mm³) were divided into groups: control, heat shock alone, KNK437 alone, and KNK437 + heat shock. KNK437 was administered intraperitoneally at 100 mg/kg 1 hour before local tumor heat shock (43°C for 45 minutes). In the KNK437 + heat shock group, tumor growth was significantly inhibited: the tumor volume on day 14 post-treatment was 285 mm³ (vs. 642 mm³ in heat shock alone group). Immunohistochemical staining of tumor tissues showed that KNK437 reduced heat-induced HSP70 expression in tumor cells. No obvious tumor regression was observed in the KNK437 alone group [2]

HT-29 cell culture and thermotolerance assay: HT-29 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a 5% CO₂ incubator. For thermotolerance induction, cells were seeded in 96-well plates (5×10³ cells/well) and cultured for 24 hours. Then, cells were treated with KNK437 (0 μM, 10 μM, 50 μM, 100 μM) for 1 hour, followed by heat shock at 43°C for 30 minutes in a water bath. After heat shock, cells were incubated at 37°C for 48 hours. Cell survival was measured by MTT assay: 20 μL of MTT solution (5 mg/mL) was added to each well, incubated for 4 hours, then 150 μL of DMSO was added to dissolve formazan crystals, and absorbance was measured at 570 nm [1]
- Western blot for HSP detection in HT-29 cells: After KNK437 and heat shock treatment, HT-29 cells were washed with cold PBS and lysed in RIPA buffer containing protease inhibitors. Cell lysates were centrifuged at 12,000×g for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined by BCA assay. Equal amounts of protein (30 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 hour at room temperature, then incubated with primary antibodies against HSP70, HSP90, and β-actin (loading control) overnight at 4°C. After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. Bands were visualized using an enhanced chemiluminescence (ECL) kit and quantified by densitometry [1]
- HSC-2 cell clonogenic assay: HSC-2 cells were seeded in 6-well plates (2×10³ cells/well) and cultured for 24 hours. Cells were treated with 50 μM KNK437 for 1 hour, then subjected to heat shock at 43°C for 45 minutes. After heat shock, cells were incubated at 37°C for 10 days to allow colony formation. Colonies were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 30 minutes, and colonies with more than 50 cells were counted. The clonogenic survival rate was calculated as (number of colonies in treatment group / number of colonies in control group) × 100% [3]
- Western blot for histone H3 methylation in HSC-2 cells: HSC-2 cells were treated with KNK437 and heat shock as described above. Cell lysates were prepared, and equal amounts of protein were separated by 12% SDS-PAGE. Membranes were probed with primary antibodies against dimethyl-histone H3 (Lys9), trimethyl-histone H3 (Lys9), and β-actin, followed by secondary antibody incubation and ECL detection. Band intensities were analyzed to quantify changes in histone methylation levels [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 Colon 26 mouse tumor model, intraperitoneal injection of 100 mg/kg (single dose) of KNK437 did not cause significant acute toxicity: no mouse deaths were observed, and during the 14-day observation period, the body weight of mice in the KNK437 monotherapy group was comparable to that in the control group (no significant weight loss) [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 benzyl lactam compound that was initially identified as an inhibitor of heat tolerance acquisition and HSP induction in human colon cancer cells, suggesting its potential as a thermosensitizer [1]. In vivo mouse xenograft models have demonstrated the inhibitory effects of KNK437 on HSP synthesis and heat tolerance, supporting its potential for translational application in improving the efficacy of clinical tumor thermotherapy [2]. In human oral squamous cell carcinoma cells, KNK437 not only inhibits HSP induction but also regulates heat-induced histone H3 methylation, suggesting it may play a role in regulating chromatin remodeling related to 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

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)

Solubility in Formulation 1: 1.67 mg/mL (6.81 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: Olive oil: 30 mg/mL

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