Antiproliferative activity assay

A sulforhodamine B (SRB) assay was used to assess the antiproliferative activity of the tested compounds [35]. Briefly, tumor cells (2.5 × 10³–5.0 × 10³ cells/well depending on cell type) were seeded into 96-well plates and cultured overnight before compound exposure. After 72 h of compound treatment, the cells were fixed with trichloroacetic acid (TCA, 10%) at 4 °C for 1 h, washed with double-distilled water five times and dried at room temperature. Then, the cells were dyed with SRB (0.4% in 1% acetic acid) at room temperature for 30 min, washed with 1% acetic acid five times, and dried at room temperature successively. Finally, 10 mmol/L Tris base solution was added, and the absorbance at 510 nm was measured in the microplate reader (EL800, Bio-TEK, USA). The data were processed as described [36]. The results were expressed as the means ± SD of at least three independent experiments. The antiproliferative activity was expressed as the inhibitory rate. The inhibitory rate (%) = (A_{control,72 h} − A_{compound,72 h})/(A_{control,72 h} − A_{control,0 h}) × 100, where A is the absorbance at 510 nm.