Figure 7. Role of serine proteases in HAMLET-induced death of tumor cells and pneumococci.

A) A549 carcinoma cells were preincubated for 10 minutes with diluent, 25 µM zVAD-fmk (pan-caspase inhibitor), 100 µM dichloroisocoumarin (serine protease inhibitor), or 100 µM calpeptin (calcium-dependent cysteine protease inhibitor) before being treated with 300 µg/ml of HAMLET. After 6 hours of incubation cell viability was measured using trypan blue exclusion. The graph depicts the mean death in % obtained after 3 individual experiments. The error bars represent the standard deviation. * and *** represent P<0.05 and P<0.001, respectively. B and C) S. pneumoniae AL2 (D39 Δ lytA) were preincubated for 10 minutes with diluent, 25 µM Aprotinin (serine protease inhibitor), 25 µM zVAD-fmk (pan-caspase inhibitor), or 10 µM E-64 (cysteine protease inhibitor) before being treated with 50 µg/ml of HAMLET. After 2 hours viability was determined and samples were analyzed for high molecular weight DNA fragmentation. B) Viability. The graph depicts the mean log₁₀ death obtained from five individual experiments. The error bars represent the standard deviation. ** represents P<0.01. C) DNA fragmentation. Untr indicates untreated bacteria. The remaining samples were treated with HAMLET in the presence of diluent (none) or proteasee inhibitors. Only the serine protease inhibitors aprotinin rescued pneumococci from death and DNA fragmentation.