3.5. MTT Assay

A MTT assay was performed to evaluate the cell viability. The MTT was obtained from Molecular Probes Inc. (Eugene, OR, USA). Briefly, the cells (1.0 × 10⁵/well) were seeded in a 96-well plate and incubated for 24 h. The cells were treated with extracts (0.125–200 μg/mL) and compounds (0.125–200 μM) and incubated for 48 h. MTT solution was added into each well (final concentration; 0.5 mg/mL) and the plates were incubated for an additional 4 h. The medium was removed and 50 μL of dimethyl sulfoxide (DMSO) was added. The optical density was measured at 540 nm using a microplate spectrophotometer (SpectraMax; Molecular Devices, Sunnyvale, CA, USA). To investigate the involvement of caspases in asarinin (1)-induced cell death, a MTT assay was also performed using caspase inhibitors. The cells were pretreated with caspase inhibitors (50 μM) for 30 min, and treated with (−)-asarinin (1) (A2780, 40 μM; and SKOV3, 60 μM) for 48 h. Caspase-3 inhibitor z-DEVD-fmk, caspase-8 inhibitor z-IETD-fmk, and caspase-9 inhibitor z-LEHD-fmk were purchased from Calbiochem (Bad Soden, Germany).