Natural flavonoid; antioxidant, anticancer, neuroprotective
Sirtuin 1 (SIRT1): Fisetin (Fustel; CCRIS-9034; NSC-407010) induces SIRT1 expression, but no IC50/Ki/EC50 values for direct binding or enzymatic modulation were reported [1]
- β-tubulin (a structural component of microtubules): Fisetin binds to β-tubulin and disrupts microtubule dynamics, but no quantitative binding parameters (e.g., Ki, KD) were provided [2]

Fisetin reduces lipid accumulation and lowers PPARγ expression in 3T3-L1 cells. Fisetin suppresses early phases of preadipocyte development and stimulates Sirt1 expression. Fisetin stimulates Sirt1-mediated deacetylation of PPARγ and FoxO1, boosts the binding of Sirt1 to the PPARγ promoter, consequently decreasing PPARγ transcriptional activity, thereby inhibiting adipogenesis [1]. Fisetin binds tubulin and stabilizes microtubules, having binding properties substantially superior to those of paclitaxel. Fisetin treatment of human prostate cancer cells resulted in substantial overexpression of microtubule-associated proteins (MAP)-2 and -4. Fisetin strongly suppresses PCa cell growth, migration and invasion. Nudc, a protein linked with the microtubule motor dynein/dynamin complex that governs microtubule dynamics, is inhibited by Fisetin therapy [2].

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In 3T3-L1 preadipocytes, Fisetin (10 μM, 20 μM) treatment during adipogenic differentiation (8 days) dose-dependently inhibited fat accumulation: Oil Red O staining showed a 35% reduction at 10 μM and 60% reduction at 20 μM vs. vehicle. Western blot revealed increased SIRT1 protein (2.0-fold at 20 μM) and decreased adipogenic markers (PPARγ: 45% reduction; C/EBPα: 50% reduction at 20 μM). qRT-PCR confirmed SIRT1 mRNA upregulation (1.8-fold at 20 μM) [1]
- In human prostate cancer cell lines (PC-3, DU145), Fisetin (10 μM–50 μM) treatment for 72 hours dose-dependently inhibited cell proliferation: IC50 = 25 μM (PC-3), IC50 = 30 μM (DU145) via MTT assay. Immunofluorescence showed disrupted microtubule structure (fragmented microtubules at 25 μM), and flow cytometry revealed increased apoptotic cells (from 5% to 38% at 25 μM in PC-3) with elevated cleaved caspase-3 (2.5-fold at 25 μM) via Western blot [2]
- In PC-3 cells, Fisetin (25 μM) blocked cell cycle progression at G2/M phase: G2/M population increased from 15% (vehicle) to 42% (25 μM) via flow cytometry, associated with reduced cyclin B1 (40% reduction) and increased p-CDK1 (2.0-fold) [2]

Treatment with fisetin decreased inflammatory cell infiltration and proliferation in mice exposed to UVB rays. Additionally, treating with fisetin lowers the levels of inflammatory mediators like COX-2, PGE2 and its receptors (EP1–EP4), and MPO activity. Moreover, fisetin lowers UVB-exposed skin's concentrations of the inflammatory cytokines TNFα, IL-1β, and IL-6. Increased expression of the proteins p53 and p21 indicates that fisetin treatment also reduces DNA damage and markers of cell proliferation [3].

In vitro microtubule polymerization assay[2]
Tubulin proteins were suspended in G-PEM buffer plus 3% glycerol in the absence (control) or presence of fisetin or paclitaxel at 10 μM concentration at 4 °C. Polymerization was followed by measuring the increase in fluorescence over a 60 minute period at 37 °C.

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Surface plasmon resonance (SPR) binding assays[2]
Binding experiments were performed using a Biacore T-200 instrument at 25 °C. Human β-tubulin full length protein (1 aa–444 aa, 76 kDa including GST tag), 6000 RU (response units) of the protein, was directly immobilized on flow cell 2 by amine coupling method using N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in water. The same number of RU of GST alone was immobilized on flow cell 1 for reference subtraction. GST-antigen was flowed over the chip (CM5, GE certified) at variable concentrations in 10 mM sodium acetate buffer (pH 4.0), with a low rate of 1 μl/min. Binding of antigen to the anti-GST antibodies was monitored in real time to obtain on (ka) and off (kd) rates. The equilibrium constant (KD) was calculated by steady state kinetics due to fast off rate. Both paclitaxel and fisetin stocks were prepared in 100% DMSO, and further dilutions were made in assay buffer containing 10 mM HEPES buffer (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% P20 (polyoxyethylenesorbitan), 1 mM CaCl2 and 5% DMSO. Scouting was performed at 4000 nM of each of the analyte. Full kinetic analysis was performed using analyte concentrations from 2000 nM to 0 (run serial dilutions, 1000, 500, 250, 125 and 0), and a flow rate of 50 μl/min.

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Cold-induced microtubule depolymerization[2]
PC-3 cells were seeded on two chamber glass slides and cultured overnight. Cells were treated with fisetin (80 μM) for 24 hours, and control cells were treated with 1% DMSO. After 2 hours of treatment, glass slides were put on ice for 0–90 minutes. Cells were fixed with 4% paraformaldehyde and 0.5% glutaraldehyde for 10 min at room temperature, and then permeabilized and saturated with a solution of 0.1% Triton X100 in phosphate buffered saline (PBS)–1% bovine serum albumin for 1 hour at room temperature. Cells were incubated overnight at 4 °C with a primary monoclonal antibody anti-α-tubulin and then incubated for 1 hour at room temperature with a mouse secondary FITC labeled antibody. Fixed cells were mounted on glass slides with antifade agent Prolong Gold-DAPI and photographed using a Nikon fluorescence microscope.

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Microtubule Polymerization Assay: Purified bovine brain tubulin was incubated with GTP (1 mM) and serial dilutions of Fisetin (10 μM–50 μM) in polymerization buffer (80 mM PIPES pH 6.9, 2 mM MgCl₂, 0.5 mM EGTA). Microtubule polymerization was monitored by measuring fluorescence intensity (excitation 350 nm, emission 440 nm) at 37°C for 60 minutes. Fisetin (25 μM) inhibited polymerization by 55% vs. vehicle, confirming microtubule disruption [2]

Western blot analysis[2]
\nPCa cells were treated with fisetin or control for 24 hours, harvested and then lysed in RIPA buffer. A total of 30 μg of protein was mixed with Laemmli sampling loading buffer, separated by 6–15% gradient SDS-PAGE. Then the samples were transferred to nitrocellulose and incubated with primary and appropriate HRP-conjugated secondary antibodies. Proteins were detected by enhanced chemiluminescence using Bio-Rad chemi-Doc MP system.[2] \n

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\nCell migration[2]
\nMigration was studied by wound-healing assay. PC-3 cells were plated in 60 mm2 Petri dish plates, when the cells ~90% confluent, a wound was induced by scraping a gap using a micropipette tip. The speed of wound closure was compared between fisetin 10–80 μM treated and untreated control groups. Photographs were taken 0–72 hours after wound incision.\n

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\nCell invasion assay[2]
\nPC-3 cells were analyzed for invasion using the Chemicon cell invasion assay kit according to the manufacturer’s protocol. Briefly, suspension of 0.5–1.0 × 106 cells/ml in serum-free medium was loaded into ECMatrex layer inserts and allowed to migrate to bottom of membrane for 72 hours at 37 °C. Cells were removed from the top of the inserts and those that invaded through the polycarbonate/basement membrane were fixed, stained and quantified by dissolving stained cells in 10% acetic acid (100–200 μl/well) and transfer a consistent amount of the dye/solute mixture to a 96 well plate for colorimetric reading of OD 560 nm.\n

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\nBrdU cell proliferation chemiluminescent assay[2]
\nPC-3 cells were analyzed for cell proliferation using BrdU cell proliferation assay kit according to the manufacturer’s protocol. Briefly, cells are cultured with labeling medium that contains BrdU; this pyrimidine analog is incorporated in place of thymidine into the newly synthesized DNA of proliferating cells. After removing the labeling medium, cells were fixed and the DNA was denatured with a fixing/denaturing solution. A BrdU mouse mAb was then added to detect the incorporated BrdU. Anti-mouse IgG, HRP-linked antibody was used to recognize the bound detection antibody. Chemiluminescent reagent was added for signal development followed by measurement of OD at 425 nm.\n

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\nCell cycle analysis by flow cytometry[2]
\nPC-3 cells were treated with or without fisetin (0–80 μM) for 24 hours. Then, cells were harvested and fixed and processed according to the manufacturer’s protocol. Stained cells were immediately analyzed by FACS Calibur for cell cycle analysis.\n

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\nViability assay[2]
\nCells were seeded (6 × 104 cells/2 mL) in 6-well plates (24 hours) and treated with or without fisetin (0–80 μM) for 24, 48, and 72 hours. Cell viability was determined by Trypan blue assay (Bio-Rad, CA) according to the manufacturer’s protocol.\n

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\nClonogenic assay[2]
\nCells were seeded (500–1000 cells/5 mL) in 60 mm2 Petri dish plates for 24 hours. Cells were treated with or without fisetin (0–80 μM) and allowed to form colonies in 1–3 weeks. Colonies were fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and photographs were taken.\n

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\nFisetin (3,7,3',4'-tetrahydroxyflavone) is a naturally found flavonol in many fruits and vegetables and is known to have anti-aging, anti-cancer and anti-viral effects. However, the effects of fisetin on early adipocyte differentiation and the epigenetic regulator controlling adipogenic transcription factors remain unclear. Here, we show that fisetin inhibits lipid accumulation and suppresses the expression of PPARγ in 3T3-L1 cells. Fisetin suppressed early stages of preadipocyte differentiation, and induced expression of Sirt1. Depletion of Sirt1 abolished the inhibitory effects of fisetin on intracellular lipid accumulation and on PPARγ expression. Mechanistically, fisetin facilitated Sirt1-mediated deacetylation of PPARγ and FoxO1, and enhanced the association of Sirt1 with the PPARγ promoter, leading to suppression of PPARγ transcriptional activity, thereby repressing adipogenesis. Lowering Sirt1 levels reversed the effects of fisetin on deacetylation of PPARγ and increased PPARγ transactivation. Collectively, our results suggest the effects of fisetin in increasing Sirt1 expression and in epigenetic control of early adipogenesis[1].

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3T3-L1 Adipogenesis Assay: 3T3-L1 preadipocytes were seeded in 24-well plates and induced to differentiate with a cocktail (insulin, dexamethasone, IBMX) plus Fisetin (10 μM, 20 μM) or vehicle for 8 days. Cells were stained with Oil Red O, and absorbance at 510 nm was measured to quantify fat accumulation. For Western blot/qRT-PCR, cells were lysed, proteins/RNA were extracted, and SIRT1, PPARγ, C/EBPα levels were detected [1]
- Prostate Cancer Cell Proliferation & Apoptosis Assay: PC-3/DU145 cells were seeded in 96-well plates (5×10³ cells/well) and treated with Fisetin (10 μM–50 μM) for 72 hours; MTT assay measured viability to calculate IC50. For apoptosis, PC-3 cells were treated with 25 μM Fisetin for 48 hours, stained with Annexin V-FITC/PI, and analyzed by flow cytometry. Western blot detected cleaved caspase-3 [2]
- Microtubule Structure & Cell Cycle Assay: PC-3 cells were treated with 25 μM Fisetin for 24 hours, fixed, permeabilized, and stained with anti-β-tubulin antibody (fluorescent secondary antibody) for immunofluorescence. For cell cycle, cells were stained with propidium iodide and analyzed by flow cytometry to quantify G2/M phase population [2]

topical treatment of Fisetin 250 nmol/0.2 mL acetone/mouse Mice: The mice are divided into six groups of eight animals each. The mice in the first group are topically treated with 0.2 mL acetone and used as vehicle control. The mice in the second and third groups receive topical treatment of Fisetin 250 nmol/0.2 mL acetone/mouse and 500 nmol/0.2 mL acetone/mouse respectively on their dorsal skin and serves as agent controls. The mice in the fourth, fifth and sixth groups are exposed to UVB. The mice in the fourth group receive a topical application of 0.2 mL acetone after 15 min of UVB exposure designated as vehicle control. The mice in the fifth and sixth groups are treated with topical application of Fisetin 250 nmol/0.2 mL acetone/mouse and 500 nmol/0.2 mL acetone/mouse respectively on to their dorsal skin after 15 min of UVB exposure

Metabolism / Metabolites
The known human metabolites of fisetin include (2S,3S,4S,5R)-6-[2-(3,4-dihydroxyphenyl)-7-hydroxy-4-oxochromen-3-yl]oxy-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid.

The intravenous LD50 in mice was 180 mg/kg. NIOSH Exchange Chemicals Laboratory, U.S. Army Armament Research and Development Command, NX#01998. In normal human foreskin fibroblasts (NHFF), fisetin (at concentrations up to 50 μM, for 72 hours) did not show significant cytotoxicity (cell viability >80%), indicating selective toxicity against cancer cells [2].

[1]. Fisetin induces Sirt1 expression while inhibiting early adipogenesis in 3T3-L1 cells. Biochem Biophys Res Commun. 2015 Nov 27;467(4):638-44.

[2]. Dietary flavonoid fisetin binds to β-tubulin and disrupts microtubule dynamics in prostate cancer cells. Cancer Lett. 2015 Oct 28;367(2):173-83.

Feselon is a 7-hydroxyflavonol with additional hydroxyl groups at the 3, 3', and 4' positions. It possesses multiple functions, including as an EC 5.99.1.3 [DNA topoisomerase (ATP hydrolysis)] inhibitor, antioxidant, anti-inflammatory agent, metabolite, plant metabolite, and anti-aging agent. It is a 3'-hydroxyflavonoid, 7-hydroxyflavonol, and tetrahydroxyflavonoid. It is the conjugate acid of feselon (1-). Feselon has been reported in soybeans, Streptomyces, and other organisms with relevant data. Feselon is a naturally occurring polyphenol with high oral bioavailability, found in many fruits and vegetables, and possesses potential antioxidant, neuroprotective, anti-inflammatory, antitumor, senescent cell scavenging, and longevity-promoting activities. As an antioxidant, feselon scavenge free radicals after administration, protects cells from oxidative stress damage, and upregulates glutathione levels. It can also inhibit pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and nuclear factor-κB (NF-κB). Fisexin promotes cell metabolism, delays cellular senescence, regulates sirtuin function, and may prolong lifespan. Furthermore, fisexin exerts its anti-cancer effects by inhibiting certain signaling pathways. It can also inhibit certain anti-apoptotic proteins and induce apoptosis in susceptible cells.

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Fustel (CCRIS-9034; NSC-407010) is a natural flavonol found in fruits (such as strawberries and apples) and vegetables, and has attracted much attention for its anti-lipogenesis and anti-cancer properties[1][2]
- In the process of adipogenesis, Fustel exerts its effect by upregulating SIRT1 expression, which inhibits the transcription of the main regulators of adipogenesis (PPARγ, C/EBPα)[1]
- In prostate cancer, Fustel inhibits cell proliferation and induces apoptosis by binding to β-tubulin, disrupting microtubule dynamics, blocking the cell cycle progression in the G2/M phase[2]

C15H10O6

286.24

286.047

C, 62.94; H, 3.52; O, 33.54

528-48-3

Fisetin quarterhydrate;Fisetin (Standard);528-48-3

5281614

Light yellow to yellow solid powder

1.7±0.1 g/cm3

599.4±50.0 °C at 760 mmHg

330ºC

233.0±23.6 °C

0.0±1.8 mmHg at 25°C

1.785

2.52

4

6

1

21

459

0

XHEFDIBZLJXQHF-UHFFFAOYSA-N

InChI=1S/C15H10O6/c16-8-2-3-9-12(6-8)21-15(14(20)13(9)19)7-1-4-10(17)11(18)5-7/h1-6,16-18,20H

2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one

2934.99.9001

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.

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.27 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 20.8 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: ≥ 2.08 mg/mL (7.27 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 20.8 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: 10 mg/mL (34.94 mM) in 45% PEG300 5% Tween-80 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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