Genistein (NPI 031L) is an isoflavonoid derived from soy products. It inhibits protein-tyrosine kinase and topoisomerase-II (DNA TOPOISOMERASES, TYPE II), with antineoplastic and antitumor activities.

Genistein exhibits a preventive effect on breast and prostate cancer, as well as other endocrine-dependent tumors, in adult animals. In mice, when combined with radiation therapy for prostate tumors, Genistein can inhibit the growth of primary tumors and control the metastasis of tumor cells to the lymph nodes adjacent to the aorta.

Genistein acts as an agonist for the GPR30 receptor and can bind to PPARγ with a dissociation constant (Ki) of 5.7 μM. In A431 epidermoid carcinoma cells, genistein inhibits tyrosine phosphorylation stimulated by EGF. Furthermore, in NIH-3T3 cells, genistein blocks mitogenic actions mediated by EGF, insulin, and thrombin.

Purified FXa is obtained after activation with Russell's viper venom followed by affinity chromatography. The resulting FXa is > 95% pure as judged by sodium dodecylsulfate polyacrylamide gel electrophoresis. The substrate affinity values for FXa, expressed as the Michaelis-Menten-Henri constant (Km), for human, rabbit, rat and dog FXa are determined using the chromogenic substrate S-2765, and are 36, 60, 240 and 70 μM, respectively. The substrate hydrolysis is monitored by measuring absorbance at 405 nm at 25°C for up to 30 min using a SpectraMax 384 Plus plate reader and SoftMax. FXa activity for each substrate and inhibitor concentration pair is determined in duplicate. The Ki values are calculated by non-linear least-squares fitting of the steady-state substrate hydrolysis rates to the equation for competitive inhibition (Equation 1) using GRAFIT, where v equals reactions velocity in OD min?1, Vmax equals maxiumum reaction velocity, S equals substrate concentration, and I equals inhibitor concentration.

Genistein is dissolved in DMSO and stored, and then diluted with appropriate medium before use[1]. The IC50 values for Genistein are determined by the MTT assay. Briefly, the MTT assay is a colorimetric assay that is based on the ability of living but not dead cells to reduce a tetrazolium-based compound to a blue formazan product. The formazan crystals are solubilized in DMSO, and the absorbance is measured at 540 nm. The absorbance at 540 nm is proportional to the number of viable cells. The lC50 values obtained with the MTT assay are compared with the lC50 values obtained by counting viable cells using trypan blue dye exclusion and by tritiated thymidine incorporation into DNA[1].