Figure 3.

Efficient killing of human melanoma A2058 cells requires the intermolecular complementation of the two groups of PrAg proteins. (a) Human melanoma A2058 cells cultured to 50% confluence were incubated with various ratios of PrAg proteins as indicated together with FP59 (1.9 nM) for 48 h, whereafter MTT was added to determine cell viability. Cytotoxicity to the tumor cells was demonstrated with a wide range of ratios (from 1:5 to 5: 1) of PrAg-U2-R200A and PrAg-L1-R178A, PrAg-U2-R200A and PrAg-L1-I210A, and PrAg-U2-R200A and PrAg-L1-K214A. (b) Susceptibility of PrAg, PrAg-U2-R200A, and PrAg-L1-I210A to furin, MT1-MMP, and uPA. PrAg proteins were incubated with the soluble forms of furin and MT1-MMP, and uPA for the times indicated, and then analyzed by SDS-PAGE. The proteins were visualized by Western blotting using an anti-PrAg antiserum (#5308). (c) The effects of the protease inhibitors on the cytotoxicity of PrAg proteins to A2058 cells. Human melanoma A2058 cells cultured to 50% confluence were pre-incubated with PAI-1 (46 nM) and TIMP-2 (0.4 μM) for 30 min, then 3.6 nM different PrAg proteins (1.8 nM each for PrAg-U2-R200A and PrAg-L1-I210A when combined) as indicated together with FP59 (1.2 nM) were added for 48 h. MTT was added to determine cell viability at 48 h. *, p < 0.05, determined by two-tailed Student's t-test.