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| ln Vitro |
Treatment with Isosilybin A (10-90 μM) for 24 and 48 hours inhibited the growth of human prostate cancer LNCaP cells in a dose- and time-dependent manner, causing a 17-52% decrease in cell number. It induced a slight increase in cell death at higher concentrations. [1]
In human prostate cancer 22Rv1 cells, Isosilybin A (10-90 μM) treatment for 24 and 48 hours caused a 20-62% decrease in cell number and a more pronounced increase in cell death (3-16% dead cells at 30-90 μM). [1] Isosilybin A induced a significant G1 phase cell cycle arrest in both LNCaP and 22Rv1 cells, accompanied by a decrease in S phase population. [1] Western blot analysis showed that Isosilybin A treatment decreased protein levels of cyclins (D1, D3, E, A) and cyclin-dependent kinases (Cdk4, Cdk2) in LNCaP cells, and caused a slight to moderate decrease in cyclin D3, E, A, and Cdk2 in 22Rv1 cells. It also decreased Cdc25A levels in both cell lines. [1] Isosilybin A treatment increased the levels of p21, p27, and p53 in both LNCaP and 22Rv1 cells. [1] Isosilybin A induced apoptotic cell death, as evidenced by a 2- to 3-fold increase in apoptotic population in LNCaP cells and a 4- to 5-fold increase in 22Rv1 cells at 60-90 μM after 48 hours (Hoechst staining). [1] Consistent with apoptosis induction, Isosilybin A treatment increased cleavage of PARP, caspase-9, and caspase-3, and decreased the level of survivin in both LNCaP and 22Rv1 cells. [1] In non-neoplastic human prostate epithelial PWR-1E cells, Isosilybin A showed minimal effects on cell growth and viability at 24 hours, but caused a 9% decrease in cell number at 90 μM after 48 hours, suggesting a selective cytotoxicity against cancer cells. [1] An earlier study cited within this review reported an IC50 of 32 μM for Isosilybin A in human prostate carcinoma DU145 cells. [1] |
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| Cell Assay |
For cell growth and death assays, LNCaP, 22Rv1, and PWR-1E cells were plated at 40-50% confluency and treated with Isosilybin A (10-90 μM, dissolved in DMSO, final DMSO ≤0.1%) in complete medium for 24 or 48 hours. After treatment, both adherent and non-adherent cells were collected by trypsinization, washed, and counted using a hemocytometer. Cell viability was determined by trypan blue exclusion. [1]
For cell cycle analysis, cells treated with Isosilybin A were collected, suspended in a saponin/PI solution containing propidium iodide and RNase A, incubated overnight at 4°C, and analyzed by flow cytometry to determine DNA content and cell cycle distribution. [1] For apoptosis analysis (Hoechst assay), cells treated with Isosilybin A were collected, stained with DNA-binding dyes Hoechst 33342 and propidium iodide, and apoptotic cells (characteristic nuclear morphology) were quantified by counting under a fluorescence microscope. [1] For western blot analysis, cells treated with Isosilybin A were lysed in a non-denaturing lysis buffer. Equal amounts of protein (50-70 μg) were separated by SDS-PAGE, transferred to nitrocellulose membranes, blocked, and probed with specific primary antibodies against target proteins (cyclins, CDKs, CDKIs, p53, cleaved caspases, PARP, survivin). Proteins were detected using peroxidase-conjugated secondary antibodies and an ECL system. Blots were stripped and reprobed with β-actin or tubulin antibodies for loading normalization. [1] |
| References |
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| Additional Infomation |
Isosilybin A is a flavonoid lignan. It is a plant metabolite, an Aspergillus metabolite, a PPARγ agonist, and an apoptosis inducer.
Isosilybin A has been reported to exist in Aspergillus iizukae, Anastatica hierochuntica, and Silybum marianum, with relevant data. See also: Silybum (partial). Isosilybin A is a diastereomer of the flavonoid lignan isolated from silybin (an extract of Silybum marianum). It accounts for approximately 5-6.5% (w/w) of silybin. [1] Its anti-prostate cancer activity mechanism involves inducing G1 phase cell cycle arrest and apoptosis. Cell cycle arrest was associated with downregulation of G1/S phase promoting proteins (cyclins D1, D3, E, A; Cdk4, Cdk2; Cdc25A) and upregulation of CDK inhibitors (p21, p27). Apoptosis was associated with increased p53 levels, caspase (-9 and -3) activation, PARP cleavage, and downregulation of the anti-apoptotic protein survivin. [1] In this study, isosilybin A was more effective than isosilybin B in inducing the expression of CDK inhibitors (p21 and p27) in 22Rv1 cells and in inducing apoptosis in both cell lines, suggesting that its pro-apoptotic effect may be stronger. However, isosilybin A was less effective than isosilybin B in inhibiting the proliferation of non-tumor PWR-1E cells. [1] This study suggests that isosilybin A and its isoform isosilybin B may have different cellular targets and efficacies due to stereochemical differences. [1] |
| Molecular Formula |
C25H22O10
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|---|---|
| Molecular Weight |
482.436
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| Exact Mass |
482.121
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| CAS # |
142796-21-2
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| PubChem CID |
11059920
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| Appearance |
White to off-white solid powder
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| Density |
1.527±0.06 g/cm3
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| Boiling Point |
793.0±60.0 °C(Predicted)
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| Melting Point |
201-203 ºC (methanol water )
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| LogP |
2.362
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
35
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| Complexity |
750
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| Defined Atom Stereocenter Count |
4
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| SMILES |
COC1=C(C=CC(=C1)[C@@H]2[C@H](OC3=C(O2)C=CC(=C3)[C@@H]4[C@H](C(=O)C5=C(C=C(C=C5O4)O)O)O)CO)O
|
| InChi Key |
FDQAOULAVFHKBX-HKTJVKLFSA-N
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| InChi Code |
InChI=1S/C25H22O10/c1-32-17-6-11(2-4-14(17)28)24-20(10-26)33-18-7-12(3-5-16(18)34-24)25-23(31)22(30)21-15(29)8-13(27)9-19(21)35-25/h2-9,20,23-29,31H,10H2,1H3/t20-,23+,24-,25-/m1/s1
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| Chemical Name |
(2R,3R)-3,5,7-trihydroxy-2-[(2R,3R)-2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-2,3-dihydrochromen-4-one
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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 |
| 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 : ~100 mg/mL (~207.28 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.18 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 (5.18 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 (5.18 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 | 2.0728 mL | 10.3640 mL | 20.7280 mL | |
| 5 mM | 0.4146 mL | 2.0728 mL | 4.1456 mL | |
| 10 mM | 0.2073 mL | 1.0364 mL | 2.0728 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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