*4.7. Cell Proliferation Assay*

The effect that GSH-NS B and D had on HCT116, HT-29, DU145, and PC-3 cell growth was evaluated by WST-1 cell proliferation assay (Roche Applied Science, Penzberg, Germany). Briefly, 2.0 × 10<sup>3</sup> HT-29, 1.5 × 10<sup>3</sup> HCT116, 5.0 × 10<sup>2</sup> DU145, and 1.2 × 10<sup>3</sup> PC-3 cells were seeded in 100 µL of growth medium in replicates (*n* = 8) in 96-well culture plates; the seeding density of each cell line

was chosen according to the best proliferation rate. The medium was removed after 24 h and the cells were incubated with in an experimental medium containing differing GSH-NS B or GSH-NS D concentrations (0.5, 1.0, 2.0, and 3.0 mg/mL). At 24, 48, and 72 h, WST-1 reagent (10 µL) was added and the plates were incubated at 37 ◦C in 5% CO<sup>2</sup> for 1.5 h. Well absorbance was measured at 450 and 620 nm (reference wavelength) on a microplate reader Asys UV 340.

Cell proliferation data were expressed as a percentage of control, that is, untreated cells. At 24, 48, and 72 h, the inhibition concentration 50% (IC50), defined as the dose of compound that inhibited 50% of cell growth, was interpolated from the growth curves, as was the inhibition concentration 1% (IC1), defined as the dose of compound that inhibited 1% of cell growth. Thus, to compare the effects of GSH-NS on the different cell lines, the IC<sup>1</sup> and IC<sup>50</sup> values obtained were used to carry out the following experiments.
