Natural flavonoid; antioxidant, anticancer, neuroprotective

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].

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.

Western blot analysis[2]
PCa 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]

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Cell migration[2]
Migration 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.

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Cell invasion assay[2]
PC-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.

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BrdU cell proliferation chemiluminescent assay[2]
PC-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.

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Cell cycle analysis by flow cytometry[2]
PC-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.

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Viability assay[2]
Cells 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.

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Clonogenic assay[2]
Cells 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.

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Fisetin (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].

Metabolism / Metabolites
Fisetin has known human metabolites that include (2S,3S,4S,5R)-6-[2-(3,4-dihydroxyphenyl)-7-hydroxy-4-oxochromen-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid.

mouse LD50 intravenous 180 mg/kg U.S. Army Armament Research & Development Command, Chemical Systems Laboratory, NIOSH Exchange Chemicals., NX#01998

[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.

Fisetin is a 7-hydroxyflavonol with additional hydroxy groups at positions 3, 3' and 4'. It has a role as an EC 5.99.1.3 [DNA topoisomerase (ATP-hydrolysing)] inhibitor, an antioxidant, an anti-inflammatory agent, a metabolite, a plant metabolite and a geroprotector. It is a 3'-hydroxyflavonoid, a 7-hydroxyflavonol and a tetrahydroxyflavone. It is a conjugate acid of a fisetin(1-).
Fisetin has been reported in Glycine max, Streptomyces, and other organisms with data available.
Fisetin is an orally bioavailable naturally occurring polyphenol found in many fruits and vegetables, with potential antioxidant, neuroprotective, anti-inflammatory, antineoplastic, senolytic, and longevity promoting activities. Upon administration, fisetin, as an antioxidant, scavenges free radicals, protect cells from oxidative stress, and is able to upregulate glutathione. It inhibits pro-inflammatory mediators, such as tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6), and nuclear factor kappa B (NF-kB). Fisetin promotes cellular metabolism, reduces senescence, regulates sirtuin function and may promote longevity. Fisetin also exerts anti-cancer activity by inhibiting certain signaling pathways. It also inhibits certain anti-apoptotic proteins and induces apoptosis in susceptible cells.

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.)