Bortezomib sensitizes murine and human RCC cells to NK-cell lysis through activation of caspase-8. RENCA tumors and human RCC cells (JOHW tumor cells) were treated with 20 nM and 10 nM bortezomib, respectively, for 18 hours, and then washed and cocultured with syngeneic murine and allogeneic human NK cells, respectively (3:1 effector-target [E/T] ratio) for an additional 5 hours and analyzed for (A,B) caspase-8 activity or (C) susceptibility to NK-cell lysis (E/T ratio = 1:1) in the presence of caspase-8 (Z-IETD)– or caspase-9 (Z-LEHD)–blocking reagents (20 μM). (D) Untreated or bortezomib-treated (20 nM bortezomib for 18 hours) RENCA cells were cocultured with syngeneic NK cells (E/T ratio = 3:1) for 5 hours and analyzed for expression of Bid. (E) Cytotoxicity of freshly isolated and expanded human NK cells against human RCC cells at E/T ratios of 15:1 and 3:1, respectively. CMA indicates treatment with CMA (20 nM); α-TRAIL, anti–mouse TRAIL antibody (N2B2; 10 μg/mL) or anti–human TRAIL (RIK-2) antibody (10 μg/mL); wt, wild-type murine NK cells; and pfp−/−, perforin-deficient murine NK cells. x-axis in panel A shows log fluorescence of caspase-8. Each data point represents a separate experiment in panel B, (horizontal bars show mean) and P values were calculated by one-way ANOVA with the Tukey multiple comparison post test.