Cytotoxicity assay

MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl tetrazolium bromide) colorimetric assay was used to determine the cytotoxicity of EASPA to both HeLa cells and PC3 according to a previously reported method [23]. The HeLa cells were seeded onto a 96-well plate at a concentration of 1 × 10⁵ cells/mL (100 μL/well) in DMEM culture medium and cultured at 37 °C for 24 h. Subsequently, the culture medium was replaced with 200 μL of fresh DMEM containing EASPA at different concentrations (5, 20, 80 μg/mL) and incubated for 48 h. The DMEM was employed as a negative control. Thereafter, 200 μL MTT solution (0.5 mg/mL) in phosphate buffered saline was added to each well, and the plate was incubated for 4 h. The solutions in each well were then removed, and the remaining formazan crystals in the cells were dissolved with 100 μL of DMSO. The absorbance of the mixture in the 96-well plate was then measured using a microplate reader at 570 nm. The cytotoxicity of EASPA at different concentrations was calculated as the percentage of the absorbance relative to that of the negative control. Tamoxifen was used as a positive control. Experiments were performed in triplicate. The results (% inhibition) were processed using Soft- Max Pro software (Molecular Device, USA).

The same procedure was adapted for PC3 cells as well. Doxorubicin was used as a positive control.