FIGURE 4:

TNF-apoptotic pathway is involved for AGP-induced selective apoptosis in cancer cells. (a, b) Mel007 cells were treated with AGP (5, 15, 30 s), and gene expression for TNFR1 was measured by quantitative real-time PCR and Western blotting. (c) Effect of AGP on the activation of TNF ligand in Mel007 and melanocytes was determined by enzyme-linked immunosorbent assay. Cells were treated with AGP (5, 15, 30 s) for 48 h. The concentration of TNFα was measured in cell culture supernatant. (d) Effect of AGP pretreated with or without TNFR1-neutralizing antibody on stress response targets were determined by Western blot analysis of H2AX and γ-H2AX protein in melanoma cells (Mel007). Mouse IgG1 isotype antibody was used as negative control. GAPDH expression was used as loading control. (e, f) Mel007 cells were treated with AGP or pretreated with anti-TNFR1 antibody, caspase inhibitor zVAD, H₂O₂-depleting-agent catalase, and nitric oxide synthesis inhibitor DPI, followed by AGP treatment. Cell viability was measured using a cell titer nonradioactive cell proliferation assay, and caspases 3/7 activity was measured by Caspase-Glo 3/7 assay. In all experiments, control cells were mock treated with He gas flow only. All values are mean ± SD of three independent experiments performed in triplicate. *p ≤ 0.01, **p ≤ 0.001; ANOVA.