NCI-H460 Cell Growth Inhibition Assay

The human tumor cell line NCI-H460 (non-small cell lung cancer) was obtained from the National Cancer Institute and maintained at 100% humidity and 5% CO₂ at 37 °C. Cell growth inhibition assays were carried out as described previously.^{20 (link)} A broth microdilution method was used to determine the growth inhibition IC₅₀ values. Briefly, ~5 × 10⁴ cells suspended in 100 μL of DMEM supplemented with 10% fetal bovine serum (FBS), 4.5 g/L glucose and L-glutamine and preserved with 1% penicillin–streptomycin were seeded in 96-well plates (Corning Inc., Corning, NY) and incubated at 37 °C in a 5% CO₂ atmosphere. The cells were incubated with 1000 μM, 333 μM, 111 μM, 37 μM, 12 μM, 4.1 μM, 1.4 μM, 0.46 μM, 0.15 μM, and 0.051 μM of compounds and H₂O as a control for 4 days.^{20 (link)} Then, an MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay (ATCC, Manassas, VA) was performed to obtain dose–response curves. The IC₅₀ values of the free BP, Mo₆Zol₂ and Mo₄Zol₂Mn(III), which had been measured in MCF-7 cells in our first studies^{21 (link),22 (link)} were re-determined here using the same conditions as for the other compounds (Table 1).