Figure 4

NaD1-mediated tumor cell killing at subacute concentrations is not caspase-dependent. (a) The ability of NaD1 to induce caspase-dependent apoptosis in MM170 and Jurkat cells was determined using the Caspase-Glo 3/7 Assay. Caspase activity expressed as bioluminescence was detected at 6, 16 and 24 h following treatment with NaD1 at 1.25, 2.5 and 5 μM. NaD1 exposed to ultraviolet (UV) radiation for 4 h was used as a control. (b) Fragmentation of chromosomal DNA over 6, 16 and 24 h was determined by agarose gel electrophoresis following 1.25 μM (MM170) or 2.5 μM (Jurkat) treatment with NaD1, or 4 h after exposure to UV radiation. Data represent two independent experiments. (c) The ability of the pancaspase inhibitor, Q-VD-OPH, to inhibit the cytotoxic activity of NaD1 towards MM170 and Jurkat cells was determined over 6, 16h and 24 h following 1.25 μM (MM170) or 2.5 μM (Jurkat) treatment with NaD1. For MM170 cells – 6 h, P=0.686; 16 h, P=0.375; 24 h, P=0.491. For Jurkat cells – 6 h, P=0.977; 16 h, P=0.092; 24 h, P=0.485, unpaired Student’s two-tailed t-test. (d) To test directly the ability of Q-VD-OPH to inhibit apoptosis, phosphatidylserine exposure as determined by FITC-Annexin V staining of Jurkat cells 4 h after exposure to UV radiation was detected via flow cytometry. P=0.011, unpaired Student’s two-tailed t-test. Data in (a), (c) and (d) are representative of at least two independent experiments, error bars represent S.E.M, n=3. *P<0.05.