Figure 4

Saposin C reduces growth inhibitory effect of etoposide and acts as an anti-apoptotic factor for prostate cancer cells. A, cells were seeded at 2000 per well in 96-well plates in their complete culture media for 3 (for PC-3 and DU-145) or 4 days (for LNCaP), treated with vehicle (DMSO), saposin C (0.1, 1, or 10 nM), prosaptide TX14A (10 nM), inactive mutant peptide 769M (10 nM), or prosaposin (1 ng/ml) at the indicated concentrations in the presence or absence of etoposide at the indicated concentrations for 3 days. After this period cell number was determined using MTS assay and cell type-specific OD/cell number calibration curve as described in Materials and Methods. B, apoptosis was determined by TUNEL assay. Cells were cultured in multiwell chamber slide up to 40% confluency in their complete culture media, and treated with etoposide in the presence or absence of saposin C (0.1, 1, or 10 nM) for 3 days. Percentage of apoptosis was determined by random selection of 10 microscopic field (at × 200 magnification) and cell count with a hemocytometer. Data expressed at the average of three independent experiments and twelve replicate samples; bars, ± SEM. * indicates P < 0.05, and ** indicates P < 0.01 compared to control (etoposide). 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.