Viability and apoptosis in KSIMM and HUVEC cultures treated with ritonavir.

Both adherent and nonadherent cells from cell cultures of KSIMM (triangles) or HUVECs (squares), treated with ritonavir at indicated concentrations for 40 hours, were harvested to determine the percentage of viable cells when compared to untreated cultures (A). SD of data points illustrated was less than 10% of the mean values as obtained from 3 different cultures per treatment concentration. Increased cell death in KSIMM cultures (▴), compared to HUVECs (▪), correlated with increased caspase-3 activity. Ritonavir-treated HUVECs differed in cell death from KSIMM (P = .015). Wilcoxon rank-sum tests were performed for all comparisons of the means. (B) Cultures were treated with ritonavir at indicated concentrations for 8 hours and enzymatic activity of caspase-3 was determined in protein extracts from cell lysates using a specific fluorogenic assay. Values represent means ± SD of triplicate determinations in relative fluorescence units (RFU). Caspase-3 values for treated HUVECs (▪) differed from KSIMM (▴; P = .015). Wilcoxon rank-sum tests were performed for all comparisons of the means. Activation of HUVECs with TNF-α increases caspase-3 activity in response to ritonavir treatment (C). Cultures were preactivated with TNF (10 ng/mL) and after 2 hours ritonavir was applied to cultures at indicated concentrations. Caspase-3 activity in activated HUVECs was measured in cell extracts after 6 hours (■) and compared with HUVECs that were not activated by TNF-α (▪). Values represent means ± SD of triplicate determinations. Caspase-3 activity in TNF-α–stimulated HUVECs differed from nonstimulated HUVECs (P = .045). Wilcoxon rank-sum tests were performed for all comparisons of the means. Four independent experiments with similar results were performed.