CYP3A4 (IC50 = 74 μM); natural flavonoid from Silybum marianum

Reporter gene detection demonstrated that silybin and isosilybin, the components of milk thistle, are responsible for suppressing PXR-mediated activation of CYP3A4 in milk thistle. Compared with silibinin, its isomer isosilybin is a greater inhibitor of PXR-mediated activation of CYP3A4. Isosilybin solutions at 89, 133 and 200 μM significantly reduced CYP3A4 induction by 64%, 82% and 88%, respectively. Isosilybin inhibits CYP3A4 induction with an IC50 of 74 μM[1]. Isosilybin B and isosilybin A are two diastereomers obtained from silymarin that possess anti-prostate cancer (PCA) action mediated through cell cycle arrest and activation of apoptosis. Treatment with isosilybin B and isosilybin A leads in growth inhibition and cell death of human prostate cancer LNCaP and 22Rv1 cells, as well as significant G(1) arrest and apoptosis [2]. Isosilybin B causes enhanced phosphorylation of Akt (Ser-473 and Thr-308) and Mdm2 (Ser-166), which is congruent with the restoration of androgen receptor levels by pretreatment with a PI3K inhibitor (LY294002). related to body degeneration. Isosilybin B therapy can accelerate the formation of complexes between Akt, Mdm2 and AR, hence boosting phosphorylation-dependent AR ubiquitination and subsequent destruction by the proteasome [3]. Isosilybin A strongly activated PPARγ at a dose of 30 μM (2.08±0.48-fold, p<0.01). Isosilybin A stimulates transactivation of the PPARγ-dependent luciferase reporter gene in a concentration-dependent manner. Computer docking studies have demonstrated that the binding manner of 3 is distinct from that of the inactive silymarin component, and there is an extra hydrogen bond with Ser342 in the entry area of the receptor ligand binding domain [4].

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The components of milk thistle responsible for this effect were identified as silybin and isosilybin. Furthermore, computational molecular docking revealed a strong interaction between both silybin and isosilybin and PXR, which was confirmed in the TR-FRET PXR assay. In conclusion, silybin and isosilybin might be suitable candidates to design potent PXR antagonists to prevent drug-drug interactions via CYP3A4 in cancer patients.[1]

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Silymarin and, one of its constituents, silibinin exert strong efficacy against prostate cancer (PCA); however, anticancer efficacy and associated mechanisms of other components of silymarin, which is a mixture of flavonolignans, are largely unknown. Here we have assessed the anticancer efficacy of two pure compounds isosilybin B and isosilybin A, isolated from silymarin, in human prostate carcinoma LNCaP and 22Rv1 cells. Isosilybin B and isosilybin A treatment resulted in growth inhibition and cell death together with a strong G(1) arrest and apoptosis in both the cell lines. In the studies examining changes in cell cycle and apoptosis regulators, isosilybin B and isosilybin A resulted in a decrease in the levels of both cyclins (D1, D3, E and A) and cyclin-dependent kinases (Cdk2, Cdk4 and cell division cycle 25A), but caused an increase in p21, p27 and p53 levels, except in 22Rv1 cells where isosilybin B caused a decrease in p21 protein level. Isosilybin B- and isosilybin A-induced apoptosis was accompanied with an increase in the cleavage of poly (ADP-ribose) polymerase, caspase-9 and caspase-3 and a decrease in survivin levels. Compared with LNCaP and 22Rv1 cells, the antiproliferative and cytotoxic potentials of isosilybin B and isosilybin A were of much lesser magnitude in non-neoplastic human prostate epithelial PWR-1E cells suggesting the transformation-selective effect of these compounds. Together, this study for the first time identified that isosilybin B and isosilybin A, two diastereoisomers isolated from silymarin, have anti-PCA activity that is mediated via cell cycle arrest and apoptosis induction.[2]

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The identification and development of novel nontoxic phytochemicals that target androgen and androgen receptor (AR) signaling remains a priority for prostate cancer (PCA) control. In the present study, we assessed the antiandrogenic efficacy of isosilybin B employing human PCA LNCaP (mutated AR), 22Rv1 (mutated AR) and LAPC4 (wild-type AR) cells. Isosilybin B (10-90 microM) treatment decreased the AR and prostate specific antigen (PSA) levels in LNCaP, 22Rv1 and LAPC4 cells, but not in non-neoplastic human prostate epithelial PWR-1E cells. Isosilybin B treatment also inhibited synthetic androgen R1881-induced nuclear localization of AR, PSA expression and cell growth, and caused G(1) arrest. In mechanistic studies identifying AR degradation, isosilybin B caused increased phosphorylation of Akt (Ser-473 and Thr-308) and Mdm2 (Ser-166), which was linked with AR degradation as pretreatment with PI3K inhibitor (LY294002)-restored AR level. Further, overexpression of kinase-dead Akt largely reversed isosilybin B mediated-AR degradation suggesting a critical role of Akt in AR degradation. Antibody pull-down results also indicated that isosilybin B treatment enhances the formation of complex between Akt, Mdm2 and AR, which promotes phosphorylation-dependent AR ubiquitination and its degradation by proteasome. Together, present findings identify a novel mechanism for isosilybin B-mediated anticancer effects in human PCA cells.[3]

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Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of glucose and lipid metabolism. Agonists of this nuclear receptor are used in the treatment of type 2 diabetes and are also studied as a potential treatment of other metabolic diseases, including nonalcoholic fatty liver disease. Silymarin, a concentrated phenolic mixture from milk thistle (Silybum marianum) seeds, is used widely as a supportive agent in the treatment of a variety of liver diseases. In this study, the PPARγ activation potential of silymarin and its main constituents was investigated. Isosilybin A (3) caused transactivation of a PPARγ-dependent luciferase reporter in a concentration-dependent manner. This effect could be reversed upon co-treatment with the PPARγ antagonist T0070907. In silico docking studies suggested a binding mode for 3 distinct from that of the inactive silymarin constituents, with one additional hydrogen bond to Ser342 in the entrance region of the ligand-binding domain of the receptor. Hence, isosilybin A (3) has been identified as the first flavonolignan PPARγ agonist, suggesting its further investigation as a modulator of this nuclear receptor.[4]

Cell culture and treatments [3]
LNCaP, 22Rv1 and PWR-1E cells were cultured as described earlier (Deep et al., 2007) and LAPC4 cells were cultured in Iscoves's modified Dulbecco's medium with 10% FBS, and 1% penicillin-streptomycin (P-S). Cells were treated with different concentrations (10–90 μM) of Isosilybin B, and/or isosilybin A, silybin A and silybin B (90 μM) in dimethyl sulfoxide (DMSO) for desired time. An equal amount of DMSO (vehicle) was present in each treatment including control, which did not exceed 0.1% (v/v). LNCaP cells were also cultured in phenol red-free medium containing 10% cFBS and 1% P-S with or without synthetic androgen R1881 for 4 days, and Isosilybin BB was then added for further 24 h. Media samples were separately collected, and cells were processed for whole-cell lysate, cytoplasmic and nuclear extracts, FACS analysis, or for cell viability assay as mentioned earlier (Muller et al., 1989; Zi et al., 1998; Zi and Agarwal, 1999).

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Western immunoblotting, and protein–protein binding and kinase assays [3]
For western blotting, 40–70 μg of protein per sample was denatured in 2 × SDS–polyacrylamide gel electrophoresis (PAGE) sample buffers and subjected to SDS–PAGE on 6, 8 or 12% polyacrylamide tris-glycine gels followed by immunoblotting as described earlier (Muller et al., 1989; Zi et al., 1998). Protein–protein binding studies were conducted as described earlier (Zi et al., 1998). Kinase activity assay for Akt was conducted as per vendor's protocol with some modifications (Dhanalakshmi et al., 2005).

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Immunofluorescence staining and confocal imaging [3]
LNCaP cells were grown in 4-chamber slides in phenol red-free medium with 10% cFBS, and were pretreated with Isosilybin B (90 μM) or DMSO for 120 min followed by synthetic androgen R1881 (1 nM) for another 150 min. Cells were then fixed in 2% paraformaldehyde overnight at 4 °C, permeabilized with 0.1% Triton X-100 for 15 min and then incubated with goat serum. Cells were washed with PBS containing 0.25% BSA and incubated with AR antibody for 1 h followed by 45 min incubation with fluorescein-tagged secondary antibody, and counterstained with DAPI for 5 min. Cell images were captured at × 400 magnification on a Nikon inverted confocal microscope using 488/402 nm laser wavelengths to detect fluorescein and DAPI emissions, respectively.

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Transfection [3]
LNCaP cells were transfected using GeneJuice as per vendor's protocol. Briefly, cells were plated in 60 mm dishes and were transfected at about 50–60% confluency where 0.5 μg of pCMV5-HA cAkt KM (kinase mutant) or pCMV5-HA (control) was incubated with GeneJuice/serum free medium mixture for 15 min and then added dropwise to the cells. Cells were treated with DMSO or Isosilybin B after 24 h of transfection, and cell lysates prepared after 24 h.

[1]. Milk thistle's active components silybin and isosilybin: novel inhibitors of PXR-mediated CYP3A4 induction. Drug Metab Dispos. 2013 Aug;41(8):1494-504.

[2]. Isosilybin B and isosilybin A inhibit growth, induce G1 arrest and cause apoptosis in human prostate cancer LNCaP and 22Rv1 cells. Carcinogenesis. 2007 Jul;28(7):1533-42.

[3]. Isosilybin B causes androgen receptor degradation in human prostate carcinoma cells via PI3K-Akt-Mdm2-mediated pathway. Oncogene. 2008 Jun 26;27(28):3986-98.

[4]. Identification of isosilybin a from milk thistle seeds as an agonist of peroxisome proliferator-activated receptor gamma. J Nat Prod. 2014 Apr 25;77(4):842-7.

Isosilybin is a flavonolignan isolated from Silybum marianum. It has a role as a plant metabolite. It is a flavonolignan, a polyphenol, a member of guaiacols and a secondary alpha-hydroxy ketone.
(2R,3R)-3,5,7-trihydroxy-2-[2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-2,3-dihydrochromen-4-one has been reported in Silybum marianum with data available.
See also: Isosilybin B (annotation moved to).

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Because cancer is often treated with combination therapy, unexpected pharmacological effects can occur because of drug-drug interactions. Several drugs are able to cause upregulation or downregulation of drug transporters or cytochrome P450 enzymes, particularly CYP3A4. Induction of CYP3A4 may result in decreased plasma levels and therapeutic efficacy of anticancer drugs. Since the pregnane X receptor (PXR) is one of the major transcriptional regulators of CYP3A4, PXR antagonists can possibly prevent CYP3A4 induction. Currently, a limited number of PXR antagonists are available. Some of these antagonists, such as sulphoraphane and coumestrol, belong to the so-called complementary and alternative medicines (CAM). Therefore, the aim was to determine the potential of selected CAM (β-carotene, Echinacea purpurea, garlic, Ginkgo biloba, ginseng, grape seed, green tea, milk thistle, saw palmetto, valerian, St. John's Wort, and vitamins B6, B12, and C) to inhibit PXR-mediated CYP3A4 induction at the transcriptional level, using a reporter gene assay and a real-time polymerase chain reaction assay in LS180 colon adenocarcinoma cells. Furthermore, computational molecular docking and a LanthaScreen time-resolved fluorescence resonance energy transfer (TR-FRET) PXR competitive binding assay were performed to explore whether the inhibiting CAM components interact with PXR. The results demonstrated that milk thistle is a strong inhibitor of PXR-mediated CYP3A4 induction. The components of milk thistle responsible for this effect were identified as silybin and isosilybin. Furthermore, computational molecular docking revealed a strong interaction between both silybin and isosilybin and PXR, which was confirmed in the TR-FRET PXR assay. In conclusion, silybin and isosilybin might be suitable candidates to design potent PXR antagonists to prevent drug-drug interactions via CYP3A4 in cancer patients.[1]

C25H22O10

482.4362

482.121

72581-71-6

Silybin A;22888-70-6;Silybin;802918-57-6;Silybin B;142797-34-0

21723007

White to off-white solid powder

1.5±0.1 g/cm3

793.0±60.0 °C at 760 mmHg

274.5±26.4 °C

0.0±2.9 mmHg at 25°C

1.684

2.59

5

10

4

35

750

2

COC1=C(C=CC(=C1)C2C(OC3=C(O2)C=CC(=C3)[C@@H]4[C@H](C(=O)C5=C(C=C(C=C5O4)O)O)O)CO)O

FDQAOULAVFHKBX-DBMPWETRSA-N

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+/m0/s1

(2R,3R)-3,5,7-trihydroxy-2-[2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-2,3-dihydrochromen-4-one

Isosilybin; 72581-71-6; 3,5,7-trihydroxy-2-[2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-2,3-dihydrochromen-4-one; 3,5,7-trihydroxy-2-[2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-3,4-dihydro-2H-1-benzopyran-4-one; 4H-1-Benzopyran-4-one, 2-(2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-1,4-benzodioxin-6-yl)-2,3-dihydro-3,5,7-trihydroxy-; CHEBI:80744; 3,5,7-trihydroxy-2-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl)-2,3-dihydrochromen-4-one; 3,5,7-trihydroxy-2-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl)-3,4-dihydro-2H-1-benzopyran-4-one;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~207.28 mM)

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.

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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.

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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.
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.

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 (Please use freshly prepared in vivo formulations for optimal results.)