| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| 5mg |
|
||
| 10mg |
|
||
| 100mg | |||
| Other Sizes |
| ln Vitro |
ψ-Bufarenogin suppresses the proliferation of seven hepatocellular carcinoma (HCC) cell lines in a dose-dependent manner. The HCC cell line SMMC-7721 was among the most sensitive. [1]
ψ-Bufarenogin inhibits cell cycle progression in HCC cells, leading to a decreased G1/S transition and a marked G2/M arrest. This was associated with reduced cyclin E expression and accumulation of cyclin B1. [1] ψ-Bufarenogin (50 nM) facilitates apoptosis in hepatoma cells after 48 hours of treatment. This pro-apoptotic effect is mediated through the downregulation of the anti-apoptotic protein Mcl-1, while Bax and Bcl-2 levels remain unchanged. Reconstitution of Mcl-1 expression attenuates ψ-Bufarenogin-triggered apoptosis. [1] ψ-Bufarenogin represses the expansion of hepatic tumor-initiating cells (T-ICs). It inhibits the expression of T-IC biomarkers (EpCAM, CD133, CD90), significantly reduces spheroid formation in HCC cell lines and primary patient cells, and diminishes the proportion of T-ICs as shown by limiting dilution assays. This effect is associated with the downregulation of the stemness-associated transcription factor Sox2. [1] ψ-Bufarenogin exhibits a synergistic effect with cisplatin in suppressing spheroid formation and in triggering apoptosis of patient-derived primary hepatoma cells. [1] ψ-Bufarenogin inhibits the activation of the Raf/MEK/ERK signaling pathway and the PI3-K/Akt cascade in HCC cells stimulated by EGF, in a dose-dependent manner. It slightly inhibits JNK phosphorylation but does not affect p38 activation. The inhibition of the PI3-K/Akt pathway is involved in the ψ-Bufarenogin-mediated reduction of Mcl-1 and Sox2. [1] ψ-Bufarenogin inhibits the auto-phosphorylation of EGFR and c-Met in HCC cells stimulated by EGF or HGF, respectively, and subsequently suppresses their downstream MEK/ERK and PI3-K/Akt signaling. [1] ψ-Bufarenogin exhibits potent cytotoxicity against hepatoma cells but has modest activity in normal hepatocytes. [1] Molecular docking studies predict that ψ-Bufarenogin binds to the kinase domains of EGFR and c-Met, forming specific hydrogen bond interactions. [1] |
|---|---|
| ln Vivo |
Intravenous (i.v.) administration of ψ-Bufarenogin (0.2 mg/kg and 0.4 mg/kg, once daily for 16 days) significantly inhibits the growth of SMMC-7721 cell-derived xenograft tumors in nude mice, reducing tumor volume by up to 60% compared to the control group, without causing obvious side effects as assessed by H&E staining of vital organs. [1]
Intratumoral injection of ψ-Bufarenogin (0.5 mg/kg and 1.0 mg/kg, every other day) also significantly inhibits xenografted HCC growth in mice. [1] Intratumoral injection of ψ-Bufarenogin (1.0 mg/kg, every other day for 24 days) inhibits the growth of a patient-derived xenograft (PDX) HCC model characterized by high expression of EGFR and c-Met. [1] Tumors from ψ-Bufarenogin-treated mice show lower Ki67 antigen expression (indicating reduced proliferation), increased TUNEL-positive cells (indicating increased apoptosis), and reduced phosphorylation of MEK and Akt compared to controls. [1] |
| Enzyme Assay |
PI3-K Kinase Activity Assay: The PI3-K enzyme was immunoprecipitated from EGF-stimulated SMMC-7721 cells pretreated with or without ψ-Bufarenogin. The kinase activity in the immunoprecipitates was measured using a competitive enzyme-linked immunosorbent assay kit according to the manufacturer's instructions. The assay demonstrated that ψ-Bufarenogin significantly represses the kinase activity of PI3-K. [1]
c-MET Tyrosine Phosphorylation Assay: SMMC-7721 cells preincubated with ψ-Bufarenogin for 4 hours were stimulated with a mitogen cocktail containing HGF. Cell lysates were extracted, and tyrosine phosphorylation of c-MET was detected using a bead-based flow cytometric assay according to the manufacturer's protocol. [1] |
| Cell Assay |
Cell Viability/Proliferation Assay (CCK-8): HCC cells were exposed to ψ-Bufarenogin at indicated concentrations for 48 hours. Cell viability was measured using a CCK-8 assay, and the inhibition rate was calculated. IC50 values were determined for different cell lines. [1]
Cell Cycle Analysis: Hepatoma cells treated with ψ-Bufarenogin were collected, fixed, stained with propidium iodide, and analyzed by flow cytometry to determine cell cycle distribution. [1] Apoptosis Assay: Apoptosis in hepatoma cells triggered by ψ-Bufarenogin was examined using a Vybrant Apoptosis Kit and analyzed by flow cytometry. [1] Anchorage-Independent Growth Assay (Soft Agar): Hepatoma cells were plated in culture dishes with medium containing Matrigel in the presence or absence of ψ-Bufarenogin. After 2 weeks, multicellular colonies were counted under a microscope. [1] Spheroid Formation Assay: Primary hepatoma cells from patients were plated in ultra-low attachment culture dishes and treated with ψ-Bufarenogin. After one week, the number of spheroids formed was counted under a microscope. [1] Limiting Dilution Assay: Hepatoma cells were seeded into 96-well ultra-low attachment plates. After 7 days, spheroid formation was assessed visually. The frequency of wells without colonies was used to estimate the proportion of stem cells using Poisson distribution statistics. [1] Western Blot Analysis: Cell lysates from treated hepatoma cells or xenograft tumor tissues were subjected to SDS-PAGE, transferred to membranes, and probed with specific primary antibodies (e.g., against p-EGFR, p-c-MET, p-MEK, p-ERK, p-Akt, Mcl-1, Sox2, cyclins). Signals were detected using IRDye-conjugated secondary antibodies. [1] Real-time PCR: Total RNA was extracted from treated cells, reverse transcribed, and amplified using gene-specific primers (e.g., for cyclin E, cyclin B1, Sox2) on a real-time PCR system to measure relative mRNA expression levels. [1] |
| Animal Protocol |
Xenograft Model (Cell Line-Derived): Fragments of SMMC-7721 xenografts were implanted subcutaneously into the flanks of nude mice. When tumors reached approximately 300 mm³, mice were randomly assigned to treatment or control groups. ψ-Bufarenogin was dissolved in DMSO and diluted with normal saline. For intravenous administration, ψ-Bufarenogin (0.2 or 0.4 mg/kg) or vehicle was injected once daily for 16 days. For intratumoral injection, ψ-Bufarenogin (0.5 or 1.0 mg/kg) or vehicle was injected every other day. Tumor dimensions were measured periodically with calipers to calculate volume. [1]
Patient-Derived Xenograft (PDX) Model: HCC tissue from a patient with high EGFR/c-MET expression was implanted into mice. When tumors were established, mice received intratumoral injections of ψ-Bufarenogin (1.0 mg/kg) or vehicle every other day for 24 days. Tumor growth was monitored as above. [1] Tissue Analysis: At the endpoint, tumors and vital organs were harvested. Parts were fixed for H&E staining, immunohistochemistry (e.g., for Ki-67, p-MEK), and TUNEL assay. Other parts were snap-frozen for protein extraction and Western blot analysis. [1] |
| Toxicity/Toxicokinetics |
In a xenograft mouse model, intravenous injection of ψ-buffalonokin at doses of 0.2 mg/kg and 0.4 mg/kg daily for 16 consecutive days did not cause significant side effects. Histopathological examination (H&E staining) of vital organs (heart, liver, spleen, lungs, and kidneys) in the treatment group mice showed no significant abnormalities compared to the control group. [1]
|
| References | |
| Additional Infomation |
ψ-Bufaranokine (C₂₄H₃₂O₆) is a bufodiene lactone compound isolated and purified from the skin of the toad (Bufo bufo gargarizans Cantor) by a two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography system. [1] It is the C-12 epimer of bufaranokine and has been reported to have nearly 100 times higher anti-cancer activity than bufaranokine. [1] Its mechanism of action is believed to be a dual inhibition of EGFR and c-MET receptor tyrosine kinases, thereby inhibiting their downstream Raf/MEK/ERK and PI3-K/Akt signaling pathways. This leads to cell proliferation arrest (through cell cycle arrest), apoptosis promotion (through downregulation of Mcl-1), and inhibition of cancer stem cell proliferation (through downregulation of Sox2). [1]
ψ-buffalokin has a synergistic effect with traditional chemotherapy drugs such as cisplatin. [1] |
| Molecular Formula |
C24H32O6
|
|---|---|
| Molecular Weight |
416.5073
|
| Exact Mass |
416.219
|
| CAS # |
17008-69-4
|
| PubChem CID |
204810
|
| Appearance |
White to off-white solid powder
|
| Density |
1.4±0.1 g/cm3
|
| Boiling Point |
635.8±55.0 °C at 760 mmHg
|
| Flash Point |
218.7±25.0 °C
|
| Vapour Pressure |
0.0±4.2 mmHg at 25°C
|
| Index of Refraction |
1.622
|
| LogP |
0.71
|
| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
6
|
| Rotatable Bond Count |
1
|
| Heavy Atom Count |
30
|
| Complexity |
847
|
| Defined Atom Stereocenter Count |
9
|
| SMILES |
C[C@]12CC[C@@H](C[C@H]1CC[C@@H]3[C@@H]2C(=O)[C@@H]([C@]4([C@@]3(CC[C@@H]4C5=COC(=O)C=C5)O)C)O)O
|
| InChi Key |
SOGONHOGEFLVPE-BHZHDSHXSA-N
|
| InChi Code |
InChI=1S/C24H32O6/c1-22-9-7-15(25)11-14(22)4-5-17-19(22)20(27)21(28)23(2)16(8-10-24(17,23)29)13-3-6-18(26)30-12-13/h3,6,12,14-17,19,21,25,28-29H,4-5,7-11H2,1-2H3/t14-,15+,16-,17-,19-,21+,22+,23+,24+/m1/s1
|
| Chemical Name |
5-[(3S,5R,8R,9S,10S,12R,13S,14S,17R)-3,12,14-trihydroxy-10,13-dimethyl-11-oxo-2,3,4,5,6,7,8,9,12,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-17-yl]pyran-2-one
|
| 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 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 (~240.09 mM)
|
|---|---|
| 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 | 2.4009 mL | 12.0045 mL | 24.0090 mL | |
| 5 mM | 0.4802 mL | 2.4009 mL | 4.8018 mL | |
| 10 mM | 0.2401 mL | 1.2005 mL | 2.4009 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
