| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| Other Sizes |
| Targets |
Musashi-1 (MSI1) (Ki = 12±2 nM against full-length MSI1; Ki = 62 nM against MSI1 RBD1)
Musashi-2 (MSI2) (Ki = 7.0±0.3 nM against full-length MSI2; Ki = 37 nM against MSI2 RRM1) Bcl-2 family (Bcl-xL Ki = 0.28 μM) [1] |
|---|---|
| ln Vitro |
In colon cancer cell lines (HCT-116, HCT-116 β/W, DLD-1), 10 μM Gn treatment led to a significant decrease in cell growth compared to controls.
Colony formation assays confirmed fewer colonies formed with higher concentrations of Gn. Gn treatment (10 μM) led to increased PARP cleavage and augmented Caspase-3 activation, indicating induction of apoptosis. At 20 μM, Gn induced autophagy in DLD-1 cells, leading to cell death via apoptosis, as shown by live-cell imaging. Gn induced autophagic flux, demonstrated by LC3 conversion and p62 degradation; p62 degradation was blocked by chloroquine (CQ) pretreatment. Gn treatment increased P21 protein and mRNA levels. Gn treatment decreased protein and mRNA levels of MSI1, c-MYC, CCND1 (CYCLIN D1), and BIRC5 (SURVIVIN). In a TOP/FOP reporter assay, Gn dose-dependently inhibited Wnt signaling reporter activity. Compared to (-)-gossypol, Gn was less effective in downregulating Notch/Wnt signaling in cells. For example, AXIN2 mRNA levels were 50% (10 μM Gn) vs. 20% (10 μM (-)-gossypol) of DMSO control in HCT-116 cells, and 80% vs. 40% in DLD-1 cells. MTT assay showed that the encapsulation of Gn using liposomes did not compromise the cytotoxicity of Gn in vitro. [1] |
| ln Vivo |
Gn-lip (Gn-loaded PEGylated liposomes) at 10 mg/kg administered via tail vein injection twice weekly for 3.5 weeks significantly inhibited the growth of human colon cancer DLD-1 xenografts in nude mice compared to the untreated control (P < 0.01, n = 10).
Mice body weight in the Gn-lip group remained stable during the treatment, indicating low systemic toxicity. Western blot analysis of tumor samples from Gn-lip treated mice showed an increase in PARP cleavage (apoptosis) and decreases in MSI1, activated Notch, CYCLIN D1, and SURVIVIN protein levels, indicating decreased Notch/Wnt signaling. [1] |
| Enzyme Assay |
A fluorescence polarization (FP) competition assay was used to screen for MSI1 inhibitors. The assay measured the ability of compounds to disrupt the binding of a fluorescein-labeled Numb RNA (5'-UAGGUAUGAGUUUUA-3') to MSI1 or MSI2 proteins. The Ki values for Gn were calculated based on the Kd and the dose-response curves.
Surface plasmon resonance (SPR) was used to confirm direct binding of Gn to MSI1-RBD1. GB1-tagged MSI1-RBD1 was immobilized on a sensor chip. At 5 μM Gn the response was 50 RU, and at 10 μM Gn the response was 200 RU, showing dose-dependent binding. Nuclear magnetic resonance (NMR) was performed to identify the binding interface. 2D 1H-15N HSQC spectra of 15N-labeled MSI1-RBD1 were titrated with Gn. The results showed that RNA-binding residues (K93, F23, and W29) were primarily affected, with changes in peak positions and decreased peak intensities. A cellular thermal shift assay (CETSA) was carried out to test drug-target engagement in cells. HCT-116 β/W cell lysates were incubated with different concentrations of Gn for 30 min, then heated at 52°C for 3 min, followed by cooling. The soluble fractions were analyzed by western blot for MSI1, showing concentration-dependent target engagement (more MSI1 protein stabilized at higher Gn concentrations). [1] |
| Cell Assay |
Cell growth was assessed by seeding cells in 96-well plates, treating with compounds, and using an MTT-based assay according to previous publications.
Colony formation assays were performed by treating cells with different doses of Gn or MP-Gr, and the number of colonies formed was counted. Apoptosis was evaluated by examining PARP cleavage via western blot and measuring Caspase-3 activation using a Caspase-3 activation assay. Autophagy was assessed using live cell imaging with an EVOS FL Auto Cell Imaging System to visualize autophagosome accumulation. Additionally, LC3 conversion and p62 degradation were analyzed by western blot. Chloroquine (CQ, 50 nM) was used to pretreat cells for 16 h to block autophagic flux. Western blot analysis was carried out to detect protein levels of MSI1, P21, c-MYC, CCND1, BIRC5, PARP, LC3, p62, and α-Tubulin. Cells were collected 48 h after drug treatment. Real-time PCR (RT-PCR and quantitative real-time PCR) was performed to measure mRNA levels of P21, AXIN2, and MSI1. Cells were collected 24 h after treatment. A TOP/FOP Wnt signaling reporter assay was carried out in HCT-116 cells treated with DMSO or different doses of Gn to evaluate Wnt inhibition. For live cell imaging, DLD-1 cells were treated with 20 μM Gn or DMSO, and images were captured over 72 hours using an EVOS FL Auto Cell Imaging System. [1] |
| Animal Protocol |
For the biodistribution study, DLD-1 tumor-bearing SCID mice were intravenously (i.v.) injected with 200 μL of DiR-loaded liposomes (10 nmol DiR) or free DiR (10 nmol in 200 μL ethanol/water 1:4 v/v). Fluorescence imaging was performed at different time points using a molecular imaging system (excitation 750 nm, emission 830 nm, exposure 60 s). Mice were euthanized at 72 h post-injection, and organs and tumors were collected for ex vivo imaging.
For the in vivo efficacy study, 5-6 week-old female athymic NCR-nu/nu nude mice were inoculated subcutaneously with 200 μL of DLD-1 cell suspension (1×10^6 cells) in plain DMEM on both flanks. When tumors reached an average volume of 40 mm^3, mice were randomized into two groups. Group 1 (10 mice, 20 tumors) was given vehicle as control. Group 2 (5 mice, 10 tumors) was given 10 mg/kg Gn-lip (Gn-loaded PEGylated liposomes). Gn-lip was administrated intravenously (i.v.) via tail vein injection twice weekly for 3.5 weeks. Tumor size (calculated as a×b^2/2, where a and b are the longest and shortest diameters) and body weight were measured twice a week. [1] |
| ADME/Pharmacokinetics |
Gn is a major metabolite of gossypol and is oxidized in the liver by P450 enzyme.
PEGylated liposomes were used to improve the bioavailability of Gn, achieve tumor-targeted delivery, and achieve controlled release of Gn, which enhanced its overall biocompatibility and drug efficacy in vivo. The biodistribution study with DiR-loaded liposomes showed that DiR signal from the liposomes increased in tumor regions over time, becoming strongest 24h after injection, and accumulated in tumors rather than the liver. In contrast, free DiR showed non-specific distribution, fast clearance, and more accumulation in the liver. [1] |
| Toxicity/Toxicokinetics |
Mice body weight in the Gn-lip treated group (10 mg/kg, i.v., twice weekly for 3.5 weeks) kept stable during the whole experimental time, indicating low systemic toxicity of the Gn-lip treatment.
Gn shares similar biological activities as gossypol, including as an inhibitor of the Bcl-2 family. [1] |
| References | |
| Additional Infomation |
Gn is a major metabolite of gossypol.
Gn was identified as a more potent inhibitor of MSI1 compared to (-)-gossypol in binding assays (Ki 12±2 nM vs 476±273 nM). Gn can potentially be used as a MSI1/2 dual inhibitor because MSI1 and MSI2 share sequence and structure similarity, especially their N-terminal RNA recognition motifs (RRMs). The working model proposes that Gn binds to the RBD1 of MSI1, disrupting MSI1-target mRNA binding, which leads to de-repression of target mRNA translation (e.g., NUMB, APC, P21), resulting in decreased Notch/Wnt signaling, decreased cell proliferation, and increased apoptosis. The limitation of Gn is that it is not specific to MSI1/MSI2, as it is also a Bcl-2 family inhibitor. This study provides a proof-of-concept for developing Gn-lip (Gn-loaded PEGylated liposomes) as a molecular therapy for colon cancer with MSI1/MSI2 overexpression. [1] |
| Molecular Formula |
C30H26O10
|
|---|---|
| Molecular Weight |
546.5214
|
| Exact Mass |
546.153
|
| CAS # |
4547-72-2
|
| PubChem CID |
197045
|
| Appearance |
Light yellow to brown solid powder
|
| Density |
1.485g/cm3
|
| Boiling Point |
842.1ºC at 760 mmHg
|
| Flash Point |
477ºC
|
| Index of Refraction |
1.702
|
| LogP |
4.472
|
| Hydrogen Bond Donor Count |
4
|
| Hydrogen Bond Acceptor Count |
10
|
| Rotatable Bond Count |
5
|
| Heavy Atom Count |
40
|
| Complexity |
1300
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
CC1=C(C(=C2C(=C(C(=C(C2=C1O)C(C)C)O)O)C=O)O)C3=C(C(=O)C4=C(C(=O)C(=O)C(=C4C3=O)C=O)C(C)C)C
|
| InChi Key |
FKZWANMBYLDTNI-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C30H26O10/c1-9(2)15-21-19(13(7-31)25(35)29(15)39)27(37)17(11(5)23(21)33)18-12(6)24(34)22-16(10(3)4)30(40)26(36)14(8-32)20(22)28(18)38/h7-10,33,35,37,39H,1-6H3
|
| Chemical Name |
7-(8-formyl-3-methyl-1,4,6,7-tetraoxo-5-propan-2-ylnaphthalen-2-yl)-2,3,5,8-tetrahydroxy-6-methyl-4-propan-2-ylnaphthalene-1-carbaldehyde
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
|
|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8298 mL | 9.1488 mL | 18.2976 mL | |
| 5 mM | 0.3660 mL | 1.8298 mL | 3.6595 mL | |
| 10 mM | 0.1830 mL | 0.9149 mL | 1.8298 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
