Cytotoxicity profiles for ProVP16-I and -II compared with VP16. The cytotoxic effect of ProVP16-I and -II was evaluated against a panel of cell lines by triplicate determinations of IC₅₀ concentrations for both prodrugs against each cell line indicated (A). The cytotoxicity mediated by the prodrugs relative to VP16 was calculated according to IC₅₀ VP16 ÷ IC₅₀ ProVP16-I or -II. Bars represent mean values ± SD. The differences between ProVP16-I or -II and VP16 were statistically significant (P < .01) for all cell lines except NXS2. Asterisks indicate cell lines with amplified MDR-1 expression. The slow release kinetics of cytotoxicity by hydrolytically activated ProVP16-I was determined using Molt-3 cells (B). In 96-well plates, 10⁴ cells per well were incubated with increasing concentrations (10^-10 M to 10^-6M) of ProVP16-I and VP16. At the time points indicated, cell viability was determined in triplicate by the XTT assay as described in “Materials and methods.” Percent cell viability was calculated from optical density measurements at 450 nm according to OD 450_sample ÷ OD 450_untreated × 100%. Results were plotted as a semilogarithmic function of drug concentration.