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Purity: ≥98%
Brusatol (also known as NSC 172924; BRN 1444847), a naturally-occuring product isolated from the Brucea javanica plant, is a novel, potent and unique inhibitor of the Nrf2 pathway that sensitizes a broad spectrum of cancer cells and A549 xenografts to cisplatin and other chemotherapeutic drugs. Mechanistically, brusatol selectively reduces the protein level of Nrf2 through enhanced ubiquitination and degradation of Nrf2. Consequently, expression of Nrf2-downstream genes is reduced and the Nrf2-dependent protective response is suppressed. In A549 xenografts, brusatol and cisplatin cotreatment induced apoptosis, reduced cell proliferation, and inhibited tumor growth more substantially when compared with cisplatin treatment alone. Additionally, A549-K xenografts, in which Nrf2 is expressed at very low levels due to ectopic expression of Keap1, do not respond to brusatol treatment, demonstrating that brusatol-mediated sensitization to cisplatin is Nrf2 dependent. Moreover, a decrease in drug detoxification and impairment in drug removal may be the primary mechanisms by which brusatol enhances the efficacy of chemotherapeutic drugs. Taken together, these results clearly demonstrate the effectiveness of using brusatol to combat chemoresistance and suggest that brusatol can be developed into an adjuvant chemotherapeutic drug. The major obstacle in cancer treatment is the resistance of cancer cells to therapies. Nrf2 is a transcription factor that regulates a cellular defense response and is ubiquitously expressed at low basal levels in normal tissues due to Keap1-dependent ubiquitination and proteasomal degradation. Recently, Nrf2 has emerged as an important contributor to chemoresistance. High constitutive expression of Nrf2 was found in many types of cancers, creating an environment conducive for cancer cell survival.
| Targets |
Nrf2 (Nuclear factor erythroid 2-related factor 2) pathway inhibitor; selectively reduces Nrf2 protein levels through enhanced ubiquitination and degradation. [1]
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| ln Vitro |
Brusatol (0.05, 0.15, 0.45, 1.35, 4.05, and 12.15μg/mL) had a dose-dependent effect on CT-26 cell viability, with an IC50 value of 0.27±0.01μg/mL. The inhibitory effect on cell growth was greatly increased when brusatol and cisplatin (CDDP) were combined at a constant concentration ratio of 1:1; the IC50 value of brusatol and CDDP co-treatment was 0.19±0.02μg /mL[2]. In mouse Hepa-1c1c7 liver cancer cells, bruscatol rapidly and transiently depletes Nrf2 protein via a post-transcriptional mechanism. The chemical toxicity of brutatol sensitizes mammalian cells [3].
Brusatol inhibits ARE-luciferase activity in a dose-dependent manner in MDA-MB-231-ARE-Luc stable cells. [1] Brusatol (40 nM) significantly decreases Nrf2 protein levels within 2–4 hours and maintains reduced levels for up to 72 hours in A549 cells. [1] Brusatol reduces protein levels of Nrf2 and its downstream genes (MRP1, MRP2, γGCS) in a dose-dependent manner in A549 cells. [1] Brusatol does not affect Keap1 protein levels. [1] Brusatol does not activate NF-κB pathway and has no effect on IκBα, phosphorylated p65, caspase-3, Stat-3, or β-catenin; moderately reduces c-Myc expression. [1] Brusatol reduces the half-life of Nrf2 from 62.4 min to 25.5 min and enhances Nrf2 ubiquitination. [1] Brusatol sensitizes A549, HeLa, and MDA-MB-231 cells to cisplatin, carboplatin, 5-fluorouracil, etoposide, and paclitaxel. [1] Brusatol (40 nM) does not induce significant apoptosis but slightly arrests cell cycle at S phase after 72 hours. [1] Brusatol decreases intracellular glutathione levels and increases intracellular cisplatin concentration. [1] |
| ln Vivo |
Brusatol can enter tumor tissue and block the Nrf2 signaling pathway. A549 cells were injected into naked mice to stimulate the growth of tumors, and then a single intraperitoneal injection of 2 mg/kg Brusatol was administered. After injection, 24 or 48 hours later, there is a significant decrease in Nrf2 protein levels [1]. Tumor size was significantly decreased in the combination group, but neither brusatol (2 mg/kg) nor cisplatin (2 mg/kg) by themselves was able to significantly slow tumor growth [1]. ?"
Brusatol (2 mg/kg, i.p.) reduces Nrf2 protein levels in A549 xenografts at 24 and 48 hours post-injection. [1] Brusatol combined with cisplatin (2 mg/kg) significantly inhibits tumor growth in A549 xenograft models, compared to cisplatin alone. [1] Brusatol enhances cisplatin-induced apoptosis and reduces cell proliferation in tumor tissues. [1] Brusatol sensitization to cisplatin is Nrf2-dependent, as shown by lack of effect in A549-K xenografts with low Nrf2 expression. [1] |
| Enzyme Assay |
ARE-luciferase reporter gene assay: MDA-MB-231-ARE-Luc stable cells were treated with brusatol for 16 hours, and luciferase activity was measured to assess Nrf2 pathway inhibition. [1]
In vivo ubiquitination assay: A549 cells were treated with brusatol (40 nM) for 4 hours, followed by immunoprecipitation and immunoblotting to detect ubiquitinated Nrf2 and Keap1. [1] |
| Cell Assay |
Cell viability assay [2]
Cell Types: mouse CT-26 CRC cell line Tested Concentrations: 0.05, 0.15, 0.45, 1.35, 4.05 and 12.15 μg/mL Incubation Duration: 48 hrs (hours) Experimental Results: The viability of CT-26 cells was dose-dependent The IC50 value is 0.27±0.01 μg/mL. Western Blot Analysis [3] Cell Types: Mouse Hepa-1c1c7 Liver Cancer Cells Tested Concentrations: 1, 3, 10, 30, 100, 300 and 1000 nM Incubation Duration: 2 hrs (hours) Experimental Results: Caused depletion of Nrf2 in a concentration-dependent manner Cells were carried out within 2 hrs (hours). Cell viability assay: A549 cells were treated with brusatol and cisplatin, and cell growth was monitored using the xCELLigence system. [1] Colony formation assay: A549 cells were treated with brusatol and cisplatin for 4 weeks, and colonies were counted. [1] Apoptosis assay: Annexin V and propidium iodide staining were used to detect apoptotic cells after brusatol treatment. [1] Cell cycle analysis: Propidium iodide staining and flow cytometry were used to analyze cell cycle distribution after brusatol treatment. [1] Western blot: Protein levels of Nrf2, Keap1, and downstream genes were analyzed after brusatol treatment. [1] qRT-PCR: mRNA levels of Nrf2 target genes were measured after brusatol treatment. [1] |
| Animal Protocol |
Animal/Disease Models: Athymic nude mice 4-6 weeks old bearing A549 xenografts [1]
Doses: 2 mg/kg Route of Administration: intraperitoneal (ip) injection treatment; cisplatin (2 mg/kg), Brusatol (2 mg/kg) , or combination therapy every other day, a total of 5 times. Experimental Results: Nrf2 protein levels diminished Dramatically 24 hrs (hrs (hours)) or 48 hrs (hrs (hours)) after injection. Cisplatin or Brusatol alone did not Dramatically inhibit tumor growth, while in the combination group, the tumor The size is Dramatically diminished.” A549 cells were injected into athymic nude mice to establish xenografts. When tumor volume reached 80 mm³ or 280 mm³, mice were treated with brusatol (2 mg/kg), cisplatin (2 mg/kg), or both via intraperitoneal injection every other day for a total of five doses. In some experiments, a second round of treatment was administered after a one-week break. Tumor volume and weight were measured, and tissues were collected for immunoblotting, TUNEL, and IHC analysis. [1] |
| Toxicity/Toxicokinetics |
Bruxol (2 mg/kg) did not cause significant weight loss or toxicity in nude mice. [1] At nanomolar concentrations, bruxol did not induce significant apoptosis or cell death in vitro. [1] Other studies have reported that higher micromolar concentrations of bruxol can inhibit protein synthesis, but this was not observed at the dose used in this study. [1]
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| References |
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| Additional Infomation |
Brusol is a triterpenoid compound. It has been reported to exist in Brucea mollis and Brucea javanica, with relevant data. Brusol is a quassinolide natural product isolated from Brucea javanica. [1] It enhances the efficacy of chemotherapy by inhibiting Nrf2-mediated defense mechanisms, thereby reducing drug detoxification and increasing intracellular drug accumulation. [1] It is considered a potential adjuvant therapy to overcome chemotherapy resistance in cancers with high Nrf2 expression. [1]
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| Molecular Formula |
C26H32O11
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|---|---|
| Molecular Weight |
520.5257
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| Exact Mass |
520.194
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| CAS # |
14907-98-3
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| PubChem CID |
73432
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| Appearance |
White to off-white solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
724.3±60.0 °C at 760 mmHg
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| Flash Point |
242.2±26.4 °C
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| Vapour Pressure |
0.0±5.3 mmHg at 25°C
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| Index of Refraction |
1.610
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| LogP |
2.08
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
37
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| Complexity |
1150
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| Defined Atom Stereocenter Count |
10
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| SMILES |
CC1=C(C(=O)C[C@]2([C@H]1C[C@@H]3[C@]45[C@@H]2[C@H]([C@@H]([C@]([C@@H]4[C@H](C(=O)O3)OC(=O)C=C(C)C)(OC5)C(=O)OC)O)O)C)O
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| InChi Key |
ZZZYHIMVKOHVIH-VILODJCFSA-N
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| InChi Code |
InChI=1S/C26H32O11/c1-10(2)6-15(28)37-18-20-25-9-35-26(20,23(33)34-5)21(31)17(30)19(25)24(4)8-13(27)16(29)11(3)12(24)7-14(25)36-22(18)32/h6,12,14,17-21,29-31H,7-9H2,1-5H3/t12-,14+,17+,18+,19+,20+,21-,24-,25+,26-/m0/s1
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| Chemical Name |
(11beta,12alpha,15beta)-13,20-Epoxy-3,11,12-trihydroxy-15-((3-methyl-1-oxo-2-butenyl)oxy)-2,16-dioxopicras-3-en-21-oic acid, methyl ester
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| Synonyms |
Brusatol; BRN 1444847; BRN-1444847; BRN1444847; NSC 172924; NSC-172924; NSC172924; (+)-Brusatol.
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| 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: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~25 mg/mL (~48.03 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.80 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 (4.80 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.80 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9211 mL | 9.6056 mL | 19.2112 mL | |
| 5 mM | 0.3842 mL | 1.9211 mL | 3.8422 mL | |
| 10 mM | 0.1921 mL | 0.9606 mL | 1.9211 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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