Figure 5

Effects of saposin C on caspase-3/7 activity (A) and the influence of PI3-kinase inhibitor (B) in etoposide-treated cells. Cell culture, treatment period, growth and caspase activity was described in Figure 4. Caspase-3/7 activity was determined using the Apo-ONE Homoheneous Caspase-3/7 assay kit based on the cleavage of a profluorescent caspase-3/7 substrate (Z-DEVD-R110) and fluorimetric quantitation was performed at an excitation and emission wavelength of 485+20 and 535+25 nm, respectively. After correction of the fluorimetric reading with the blank (vehicle control), final fluorescent intensity was depicted as an arbitrary endpoint relative fluorescent unit, RFLU. PI3-kinase inhibitor (LY294002; LY) was used at final 1.5 μM concentration and saposin C was added at optimal 1 nM (for PC-3 and LNCaP) or 10 nM (for DU-145) concenration. Etoposide (Et) was added at optimal 20 μM (for PC-3 and LNCaP) or 2 μM (for DU-145). Data expressed are the average of three independent experiments and twelve replicate samples; bars, ± SEM. * indicates P < 0.05, and ** indicates P < 0.01. Statistical significance of the effect of saposin C on cell growth and apoptosis was evaluated by one-way ANOVA with Bonferroni's corrections. Differences of vehicle (or etoposide)-only-treated cells and any other single experimental group of interest (TX14A or prosaposin) was evaluated by Student's t-test and statistical significance was set at P < 0.05.