Figure 3

Mechanisms of taurolidine-induced cytotoxicity are cell line and concentration dependent. Binding of annexin-V-biotin-FITC and propidium iodide (PI) to taurolidine-treated D17 OS cells was determined by 2-color flow cytometry. A) Data are displayed as dot plots and data points in the lower right quadrant (i.e., annexin-V positive) represent early apoptotic cells while data points in the upper left quadrant represent necrotic cells and points in the upper right quadrant (dual annexin-V/PI positive) represent cells in late apoptosis and necrosis. B) The release of nucleosomes as an indicator of apoptosis following taurolidine treatment of D17 OS cells was determined at two time points. Data are expressed as percent of apoptosis values measured in untreated cells at the same time points. The higher taurolidine concentration (250 μM) was less effective at inducing apoptotic changes in this assay indicating cell death occurred by other mechanisms. T125, T250 = 125 μM and 250 μM taurolidine respectively. * indicates different from 125 μM at same time point (p < .01). Error bars indicate SD. C) Western blots show degradation of PARP as an indirect indicator of taurolidine-activated caspase activity in COS and HMPOS cells. Cells incubated 12 hrs in taurolidine as described in methods. No loading control was available for the D17 blot because it was an immunoprecipitation. D) These proliferation assays show the caspase inhibitor (CI) Z-VAD-FMK protects HMPOS but not D17 OSA cells from caspase-dependent cell death at low concentrations of taurolidine, consistent with PARP cleavage seen in 3C. * indicates intercept is different between treated and untreated conditions (P < .05).