incubated for 18 h at 37°C. The reaction was stopped by adding CH CN. Then the mixture was lyophilized and analyzed by
3
HPLC.
Analytical HPLC was performed on anAgilent Technologies chromatograph (Series 1100) equipped with a QuatPump
G1311A high-pressure pump system, G1322A Degasser, G1328B injector, and G1314A VWD detector. The analysis results
®
were processed using ChemStation software ver. 09 (Germany). We used the literature method [4] and dihydroquercetin
(DHQ) as an internal standard.
–3
Inhibition of ꢀ-D-Glucosidase G-II by Formononetin. Enzyme solution (20 ꢃL, 10 U) in sodium succinate
buffer (0.025 M, pH 5.2) was treated with inhibitor solution (10 ꢃL) containing from 0.5 to 200 ꢃg of compound in sodium
succinate buffer (0.025 M, pH 5.2). The mixture was held at 37°C for 10 min, treated with p-nitrophenyl-ꢀ-D-glucopyranoside
(200 ꢃL, 1 mg/mL) in sodium succinate buffer (pH 5.2), and incubated at 37°C for 30 min. The residual activity was determined.
Enzymatic Synthesis of Glucosides by ꢀ-D-Glucosidase G-II. The appropriate aglycon (1 mg) and p-nitrophenyl-
–5
ꢀ-D-glucopyranoside (5 mg) were dissolved in enzyme solution (1 mL, 5 ꢄ 10 U) and incubated at 37°C for 20, 40, and
120 min and 24 h. Production of the glycosides was monitored by HPLC.
Cell Cultivation. HCT-116 human colon cancer cells (ATCC No. CCL-247) and MDA-MB-231 human breast
cancer cells (ATCC No. HTB-26) were cultivated in McCoy 5a and DMEM (Dulbecco’s modified Eagle’s medium), respectively,
and 10% phosphate buffer (FBS) with added penicillin (100 U/L) and streptomycin (100 ꢃg/L) in an MCO-18AIC incubator
(Sanyo, Japan) at 37°C with 5% CO content.
2
5
Determination of Isoflavonoid Cytotoxicity. HCT-116 and MDA-MB-231 cells (1.5 ꢄ 10 /mL) were inoculated
into 96-well plates and cultivated in 10% McCoy 5a and DMEM (200 ꢃL), respectively, in a CO incubator at 37°C for 24 h.
2
Then, cells were treated with isoflavonoids at concentrations of 3, 5, 7, 14, and 28 ꢃg/mL in fresh medium and incubated for
24 h. Each well was treated after incubation with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium (MTS reagent) (15 ꢃL) and incubated for 4 h (37°C, 5% CO ). The optical density was measured on a Bio-Tek
2
Instruments spectrophotometer (USA) at wavelength 490/630 nm (A
).
490/630
Neoplastic Cell Transformation (Soft Agar Method). The effect of the isoflavonoids on the formation and growth
of colonies of MDA-MB-231 human breast cancer cells was determined by the soft agar method [6]. MDA-MB-231 cells
4
(2.4 ꢄ 10 /mL) were treated with isoflavonoids (28 ꢃg/mL) in agar (1 mL, 0.33%) containing BME (1.8%), containing 10%
over agar (3.5 mL, 0.5%) with BME (1.8%), and containing FBS (10%) and isoflavonoid (28 ꢃg/mL). Controls were treated
with the corresponding amount of DMSO. Cells were cultivated at 37°C and 5% CO for 21 d. Human colon cancer cell
2
colonies were estimated using a Motic AE20 inverted microscope (China) and Motic Images Plus software.
Statistical data treatment was carried out using the Student t-criterion with 95% probability limits (SigmaPlot
2000, version 6, SPSS Inc., USA).
ACKNOWLEDGMENT
The work was supported by RFBR grant 09-04-00761, RAS Presidium Program Molecular and Cellular Biology, and
Presidential Grant MK 1472.2009.4.
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