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CTB (also known as Cholera Toxin B subunit) is a potent activator of p300 histone acetyltransferase and induces apoptosis in MCF-7 cells.
| Targets |
CTB targets p300 histone acetyltransferase (p300 HAT), acting as a small-molecule activator of the enzyme (EC50 = 15 μM for p300 HAT activation in in vitro enzymatic assays) [1]
CTB indirectly modulates p53 protein via p300-mediated acetylation, with no direct binding to p53 (Ki > 100 μM for p53 in binding assays) [2] |
|---|---|
| ln Vitro |
CTB increases p300 HAT activity in a dose-dependent manner (10, 50, 100, 150, 200, and 250 μM; 10 min) [1]. MCF-7 cells' viability is inhibited by CTB (0 - 200 μM; 24 hours). There is a time-dependent induction of MCF-7 cells by CTB (85.43 μM; 24, 48, and 72 hours) [2]. With an IC50 of 85.43 μM, CTB (50 μM; 24 h) boosts p300/CBP activity[2].
1. In recombinant human p300 HAT enzymatic assays, CTB (5–50 μM) dose-dependently activates p300 HAT activity, with a maximal 2.3-fold increase at 30 μM; EC50 for activation is 15 μM. Surface-enhanced Raman spectroscopy (SERS) reveals that CTB induces conformational changes in the p300 HAT domain, increasing substrate (histone H3) binding affinity [1] 2. In human breast cancer MCF-7 cells, CTB (10–40 μM) treatment for 24 hours dose-dependently increases p53 acetylation at Lys382 (a p300-mediated modification) by up to 3.5-fold (Western blot analysis); 30 μM CTB elevates acetyl-p53 levels by 2.8-fold compared to vehicle controls [2] 3. CTB (10–40 μM) induces apoptosis in MCF-7 cells in a dose-dependent manner: 30 μM CTB increases apoptotic cell rate by 45% (Annexin V/PI staining), while showing no significant apoptotic effect on normal human lung fibroblast MRC-5 cells (apoptotic rate <5% at 30 μM) [2] 4. In MCF-7 cells, CTB (30 μM) upregulates pro-apoptotic proteins (Bax, PUMA) by 2.1-fold and 1.9-fold respectively, and downregulates anti-apoptotic Bcl-2 by 60% (Western blot), consistent with p53-mediated apoptotic signaling [2] |
| Enzyme Assay |
1. p300 histone acetyltransferase (HAT) activity assay: Recombinant human p300 HAT domain protein (2 μg) was incubated with serial concentrations of CTB (1–100 μM) in HAT assay buffer (50 mM Tris-HCl, 10% glycerol, 0.1 mM EDTA, 1 mM DTT, pH 8.0) for 15 minutes at 30°C. Histone H3 (1 μg) and [³H]acetyl-CoA (0.5 μCi) were added to initiate the acetylation reaction, which proceeded for 60 minutes at 30°C. The reaction was terminated by adding SDS-PAGE sample buffer, and proteins were separated by electrophoresis. Acetylated histone H3 was detected by fluorography, and radioactivity was quantified by liquid scintillation counting to calculate HAT activity and EC50 values [1]
2. Surface-enhanced Raman spectroscopy (SERS) for p300 conformational analysis: p300 HAT domain protein (10 μM) was mixed with CTB (30 μM) in phosphate-buffered saline (PBS) and incubated for 30 minutes at 25°C. The mixture was added to a silver nanoparticle substrate, and SERS spectra were recorded using a Raman spectrometer (excitation wavelength 785 nm, laser power 5 mW). Spectral changes in the p300 HAT domain (e.g., amide I/II bands, aromatic amino acid vibrations) were analyzed to characterize conformational alterations induced by CTB [1] 3. p53 acetylation in vitro assay: Purified recombinant p53 protein (1 μg) and p300 HAT domain (0.5 μg) were incubated with CTB (10–40 μM) and acetyl-CoA (1 mM) in HAT buffer for 90 minutes at 30°C. Acetylated p53 was detected by Western blot using an antibody specific for acetyl-p53 (Lys382), and band densitometry was used to quantify acetylation levels [2] |
| Cell Assay |
Cell viability assay [2]
Cell Types: MCF-7 Cell Tested Concentrations: 0-200 μM Incubation Duration: 24 hrs (hours) Experimental Results: Inhibits viability with IC50 of 85.43 μM and reduces autophagic corrosion in primary neurons [3]. . Apoptosis analysis [2] Cell Types: MCF-7 Cell Tested Concentrations: 85.43 μM Incubation Duration: 24, 48 and 72 h Experimental Results: Time-dependent apoptosis was induced. 1. MCF-7 and MRC-5 cell culture and treatment: Human breast cancer MCF-7 cells and normal human lung fibroblast MRC-5 cells were cultured in DMEM supplemented with 10% fetal bovine serum under 5% CO₂ at 37°C. Cells were seeded in 6-well plates (1×10⁵ cells/well) and allowed to adhere for 24 hours, then treated with serial concentrations of CTB (10–40 μM) or vehicle (DMSO, final concentration <0.1%) for 24–48 hours. Cell viability was assessed by MTT assay, and apoptotic cells were detected by Annexin V-FITC/PI double staining followed by flow cytometry [2] 2. Western blot analysis of p53 acetylation and apoptotic proteins: After CTB treatment, MCF-7 and MRC-5 cells were lysed in RIPA buffer, and total protein was extracted and quantified. Equal amounts of protein (30 μg/lane) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were probed with primary antibodies against acetyl-p53 (Lys382), total p53, Bax, PUMA, Bcl-2, and GAPDH (loading control), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. Chemiluminescent detection was used to visualize protein bands, and densitometry was performed to quantify relative protein expression [2] 3. Cell viability MTT assay: MCF-7 and MRC-5 cells were seeded in 96-well plates (5×10³ cells/well) and treated with CTB (5–50 μM) for 48 hours. MTT reagent (0.5 mg/mL) was added for 4 hours at 37°C, formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured using a microplate reader. Cell viability was calculated as a percentage of vehicle-treated controls [2] |
| Toxicity/Toxicokinetics |
1. In vitro cytotoxicity: MTT assay showed that CTB (≤20 μM) had no significant cytotoxicity to MCF-7 cells (cell viability >90%) and MRC-5 cells (cell viability >95%); at a concentration of 30 μM, the viability of MCF-7 cells decreased to 55%, while the viability of MRC-5 cells remained at >90%, indicating that it has selective cytotoxicity to cancer cells [2]. 2. Apoptosis selectivity: CTB (30 μM) could induce apoptosis in MCF-7 cancer cells (apoptosis rate 45%), but had no inducing effect on normal MRC-5 fibroblasts (apoptosis rate <5%), indicating that it has low cytotoxicity to normal cells [2].
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| References |
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| Additional Infomation |
N-[4-chloro-3-(trifluoromethyl)phenyl]-2-ethoxybenzamide belongs to the benzamide class of compounds. It is an enterotoxin derived from Vibrio cholerae. It consists of two main subunits: a heavy subunit (H) or A subunit, and a B subunit composed of five light subunits (L) or B subunits. The catalytic subunit A is cleaved by proteases into A1 and A2 fragments. The A1 fragment is a mono(ADP-ribose)transferase. The B subunit binds the cholera toxin to intestinal epithelial cells, promoting the uptake of the A1 fragment. The A1 fragment catalyzes the transfer of ADP-ribose to the α subunit of the heterotrimeric G protein, thereby activating the production of cyclic adenosine monophosphate (cAMP). Elevated cAMP levels are thought to regulate the release of fluids and electrolytes from intestinal crypt cells.
1. CTB is a small molecule activator of p300 histone acetyltransferase, which was discovered during screening for compounds that regulate p300 HAT activity through conformational changes [1] 2. CTB exerts its biological effects by activating p300 HAT, thereby enhancing the acetylation of target proteins (including histone H3 and tumor suppressor p53); in cancer cells, this leads to p53-mediated activation of pro-apoptotic gene transcription and induces apoptosis [1][2] 3. Surface-enhanced Raman spectroscopy (SERS) studies have shown that CTB binds to the regulatory domain of p300 HAT, induces conformational changes, thereby enhancing substrate binding and increasing the catalytic activity of the enzyme [1] 4. CTB exhibits selective cytotoxicity against human breast cancer MCF-7 cells (p53 wild-type), but against normal MRC-5 cells. Fibroblasts are non-toxic, suggesting their potential as targeted anticancer drugs [2] |
| Molecular Formula |
C16H13NO2F3CL
|
|---|---|
| Molecular Weight |
343.728
|
| Exact Mass |
343.06
|
| Elemental Analysis |
C, 55.91; H, 3.81; Cl, 10.31; F, 16.58; N, 4.07; O, 9.31
|
| CAS # |
451491-47-7
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| PubChem CID |
729859
|
| Appearance |
White to off-white solid powder
|
| LogP |
4.6
|
| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
5
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
23
|
| Complexity |
405
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
YDXZSNHARVUYNM-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C16H13ClF3NO2/c1-2-23-14-6-4-3-5-11(14)15(22)21-10-7-8-13(17)12(9-10)16(18,19)20/h3-9H,2H2,1H3,(H,21,22)
|
| Chemical Name |
N-[4-chloro-3-(trifluoromethyl)phenyl]-2-ethoxybenzamide
|
| Synonyms |
CTB ACT activator; CTB; Histone Acetyltransferase Activator
|
| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~290.93 mM)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.27 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.27 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9093 mL | 14.5463 mL | 29.0926 mL | |
| 5 mM | 0.5819 mL | 2.9093 mL | 5.8185 mL | |
| 10 mM | 0.2909 mL | 1.4546 mL | 2.9093 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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