Cytotoxicity Screening of Cardiac Glycosides

The cytotoxicity of the cardiac glycosides was screened against the human cancer HT-29, MDA-MB-231, MDA-MB-435, or OVCAR3 cell lines, with a procedure reported previously,^{27 (link),28 (link)} with the vehicle and paclitaxel used as the negative and positive control, respectively. Briefly, after log-phase-growth, cells were seeded in 96-well clear flat-bottomed plates (Microtest 96, Falcon) and treated with a test sample or paclitaxel (both dissolved in DMSO and diluted to different concentrations required) or the vehicle (DMSO) for 72 h. Viability of cells was evaluated using a commercial absorbance assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega Corp, Madison, WI, USA), with the IC₅₀ values calculated from the vehicle control. For cytotoxicity testing against the H1299 human non-small cell lung cancer cell line, cell proliferation was assessed using the MTT proliferation assay kit (Cayman Chemical, Ann Arbor, MI, USA). Cells were seeded in each well of a 96-well plate and treated with the samples for 24 h followed by a treatment with MTT for 4 h. The medium was removed, and 100 μL Crystal Dissolving Solution were added to each well. The absorbance of the solution was measured at 570 nm, with IC₅₀ values calculated from the vehicle control.