Bax protein (required for apoptotic effect
Bcl-xL (decrease in Bcl-xL protein levels and decrease in association of Bcl-xL to Bax)
Survivin (down-regulation)
XIAP (down-regulation) [1]

Flavokawain A inhibited proliferation of RT4, T24, and EJ bladder cancer cells in a dose-dependent manner. IC50 values: T24 cells 16.7 μmol/L; EJ cells 17.2 μmol/L; RT4 cells 20.8 μmol/L. At 25 μg/mL, it caused 80-95% inhibition of proliferation (P<0.0001). [1]
Flavokawain A induced typical apoptotic morphologies including cell shrinkage, rounding up, membrane blebbing, nuclear fragmentation and condensation. [1]
Flavokawain A (12.5 μg/mL) caused activation of caspase-9 and caspase-3 in a time- and dose-dependent manner, as indicated by reduction in proenzyme intensity and appearance of cleaved fragments, and cleavage of PARP. [1]
Flavokawain A (12.5 μg/mL) induced loss of mitochondrial membrane potential: after 16 h, 11.8% of cells emitted only green fluorescence (depolarized membranes) vs 0.3% in control; after 24 h, 21.1% (P<0.01). [1]
Flavokawain A (12.5 μg/mL) caused significant release of cytochrome c from mitochondria into cytosol at 16 h. [1]
Flavokawain A (12.5 μg/mL, 8-24 h) increased Bax protein level by 54-88%, decreased Bcl-xL protein levels by 41-71%, and increased Bax/Bcl-xL ratio by 59-650%. [1]
Flavokawain A (12.5 μg/mL, 8-24 h) completely inhibited formation of Bax and Bcl-xL immunocomplexes and increased active Bax protein (recognized by anti-Bax 6A7 antibody) as early as 8 h (2- to 3-fold increase). [1]
Flavokawain A (12.5 μg/mL) down-regulated survivin (complete inhibition at 4 h) and XIAP (complete inhibition at 16 h) in a dose- and time-dependent manner. [1]
Flavokawain A inhibited anchorage-independent growth of EJ cells in soft agar in a dose-dependent manner: at 5, 12.5, and 25 μg/mL, inhibition of colony formation was 44.9%, 75.6%, and 86.9% respectively (P<0.05-0.01); at 50 μg/mL, complete inhibition. [1]

Flavokawain A (oral administration at 50 mg/kg daily for 25 days) suppressed tumor growth in a nude mouse xenograft model bearing EJ bladder tumor cells. Tumor growth curve showed decreased rate of growth compared to control. Wet tumor weights: control 465±217 mg, treatment 199±110 mg (P<0.001), representing 57% inhibition of tumor growth. [1]

Cell viability was measured by MTT assay. Cells were plated at 5×10^4 per well in 24-well plates in medium with 10% FBS. After 24 h, medium was refreshed and cells were treated as indicated. After treatment, MTT was added at 1 mg/mL and incubated at 37°C for 3 h, then absorbance determined at 570 nm. [1]
Apoptosis was assessed by Hoechst 33258 staining. Cells were treated for 24 h, fixed with 4% paraformaldehyde for 30 min at room temperature, stained with 50 ng/mL Hoechst 33258 in PBS for 30 min, then examined by fluorescence microscopy for nuclear condensation and fragmentation. [1]
Flow cytometry for apoptosis: cells were stained with FITC-conjugated Annexin V and propidium iodide in PBS. Ten thousand events collected per sample. [1]
Mitochondrial membrane potential: cells were stained with 3 μmol/L JC-1 in HBSS for 45 min at 37°C, washed, and analyzed by flow cytometry. [1]
Cytochrome c release: mitochondria and cytosol were separated using a cytochrome c-releasing apoptosis assay kit. Cells were suspended in cytosol extraction buffer, incubated on ice for 10 min, homogenized by Dounce homogenizer, centrifuged at 700×g for 10 min; supernatant centrifuged at 10,000×g for 30 min at 4°C; resulting supernatant (cytosolic fraction) and pellet (mitochondrial fraction) processed for Western blot. [1]
Western blotting: cells lysed in radioimmunoprecipitation assay buffer or CHAPS buffer (150 mmol/L NaCl, 10 mmol/L HEPES pH 7.4, 1.0% CHAPS). Clarified protein lysates (20-80 μg) resolved on SDS-PAGE (8-16%), transferred to nitrocellulose, probed with primary antibodies, detected with HRP-conjugated secondary antibodies and chemiluminescence. [1]
Immunoprecipitation: lysates (200 μg/mL) precleared with protein G plus-agarose, then precipitated with 2 μg anti-Bax 6A7 antibody and protein G plus-agarose overnight at 4°C; Bax/Bcl-xL complexes detected by immunoblotting. [1]
Soft agar colony formation: six-well plates with bottom layer of 0.5% agar in complete medium, feeder layer of 0.38% agar with 3,000 cells per well, top layer of 0.38% agar with vehicle or Flavokawain A. Cultures maintained for 14 days; colonies (>10 cells) counted under inverted phase-contrast microscope at ×100 magnification. [1]

Flavokawain A was formulated in 10% grain alcohol in 0.9% saline and given by gavage. NCR-nu/nu (nude) mice were used. EJ bladder tumor cells (2×10^6 per 200 μL) were injected subcutaneously into the right flank of each mouse. The next day, mice were randomly divided into treatment and control groups (18 mice each). Daily dosing began with vehicle or 50 mg/kg Flavokawain A for 25 days. Body weight, diet, and water consumption were recorded three times weekly. Tumor sizes measured every other day; tumor volume calculated by formula: 0.5236 × L1 × (L2)^2 (L1 = long axis, L2 = short axis). At the end, tumors were excised and weighed. [1]

Body weight gain, diet, and water consumption of Flavokawain A-treated mice were similar to control group. Mice did not show any gross abnormalities on necropsy at the end of treatment. The dose of 50 mg/kg/day was chosen as 1/60 of the LD50 (3000 mg/kg) of a similar chemical structure compound, isoliquiritigenin. [1]

[1]. Flavokawain A, a novel chalcone from kava extract, induces apoptosis in bladder cancer cells by involvement of Bax protein-dependent and mitochondria-dependent apoptotic pathway and suppresses tumor growth in mice. Cancer Res. 2005 Apr 15;65(8):3479-86.

Flavokawain A belongs to the chalcone class of compounds. 2'-Hydroxy-4,4',6'-Trimethoxychalcone has been reported in turmeric (Boesenbergia rotunda), Vitex quinata, and other organisms with available data. See also: Roots (parts) of Piper methysticum.

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Flavokawain A is a chalcone that constitutes up to 0.46% of kava extracts. Chalcones have α,β-unsaturated ketones, giving preferential reactivity toward thiols and susceptibility to Michael reaction. [1]
Consumption of traditional kava preparation correlates with low cancer incidences in kava-drinking countries (Fiji, Vanuatu, Western Samoa). Flavokawain A is identified as an active compound for bladder cancer prevention. [1]
Mechanism: Flavokawain A induces apoptosis via Bax protein-dependent and mitochondria-dependent pathway, down-regulates XIAP and survivin, and changes balance between apoptotic and anti-apoptotic molecules. The apoptotic effect is caspase-9/3-mediated and is attenuated by Bax inhibitor peptide. [1]

C18H18O5

314.3325

314.115

3420-72-2

(E)-Flavokawain A; 37951-13-6

5355469

Light yellow to yellow solid

1.2±0.1 g/cm3

529.9±50.0 °C at 760 mmHg

113 °C

193.2±23.6 °C

0.0±1.4 mmHg at 25°C

1.600

3.96

1

5

6

23

400

0

O(C([H])([H])[H])C1=C([H])C(=C([H])C(=C1C(/C(/[H])=C(\[H])/C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])[H])=O)O[H])OC([H])([H])[H]

CGIBCVBDFUTMPT-RMKNXTFCSA-N

InChI=1S/C18H18O5/c1-21-13-7-4-12(5-8-13)6-9-15(19)18-16(20)10-14(22-2)11-17(18)23-3/h4-11,20H,1-3H3/b9-6+

(E)-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-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 (~318.14 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.95 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.)