Proteasome inhibitors block actinomycin D–induced apoptosis of MM cell lines.

(A) The 8226 cells were cultured in complete medium plus IL-6 with or without the addition of actinomycin D (1 μg/mL). Indicated cultures were preincubated for 30 minutes with proteasome inhibitor MG115 or MG132 at 10 or 20 μM. After 6 hours, TUNEL assays were performed and the survival percentage was determined. (B) Cultures were prepared as in panel A, except that DRB (0.1 mM) was added instead of actinomycin D. (C) Actinomycin D–induced caspase activation is prevented by proteasome inhibitors. The 8226 cells were cultured in complete medium plus IL-6 with the addition of the proteasome inhibitor MG115 (10 μM) and /or actinomycin D, as indicated. Cells were cultured for 2 hours (lanes 1-3), 4 hours (lanes 4-6), or 6 hours (lanes 7-9), and caspase 3 activity was determined. In lane 10, an aliquot of protein extract from lane 7 was incubated with zVAD-fmk (25 μM final) immediately prior to caspase assay; caspase activity was inhibited as expected. In lane 11, MG115 (10 μM) was added to lane 7 extracts just prior to caspase assay to demonstrate that MG115 is not a direct inhibitor of caspase activity.