Enzymatic Activity Assay Protocol

MDA-MB-468 and MCF-7 tumor cells were treated with protodioscin or dioscin compounds for 24 and 48 hours, respectively. Then, after washing once with PBS (10 mM, pH 7.4), the cells were harvested and centrifuged at 1,200 g for 10 min. The pellet was suspended in 500 μL of lysis buffer composed of 50 mM Tris-HCl, 1mM phenylmethanesulfonyl (PMSF), 0.1% (v/v) Triton X-100, in 1.5 mL Eppendorf tubes and maintained in constant agitation at 4°C for 30 min. The homogenate was centrifuged (1,600 g, 20 min) at 4°C. The supernatant (enzyme extract solution) was kept at −80°C or used for the determination of Superoxide dismutase (SOD), Glutathione Peroxidase (GPx), Thioredoxin reductase (TrxR), glutathione reductase (GR), and Isocitrate dehydrogenase (NADP+-ICDH) activities.