IPSI-001 preferentially inhibits the proteasome and induces death in lymphoid-derived cells. (A) The ChT-L activity was measured in vitro in solid tumor (SK-N-MC, Hs294T, BT474, HeLa, and HPAC; lanes 1-5) and hematologic cancer cells (CCRF-CEM, U-937, ANBL-6, RPMI 8226, MOLT-4, AML193, and SupB8; lanes 6-12) by exposing lysates to 50 μM IPSI-001 or 10 nM bortezomib for 15 minutes, followed by incubation with the specific ChT-L activity substrate. The data shown are the mean plus or minus SD from experiments performed in triplicate. (B) HPAC pancreatic adenocarcinoma cells, Hs294T melanoma cells, and ANBL-6 and RPMI 8226 human myeloma cells were exposed to vehicle, 10 nM bortezomib, 25 μM E64d, or 50 μM IPSI-001 for 24 hours. Apoptosis was evaluated using an enzyme-linked immunosorbent assay that detects apoptotic nuclear DNA fragmentation. Cell death was expressed as the fold increase in apoptosis over the vehicle control, which was arbitrarily set at 1.0. Representative data are shown from 3 independent experiments. *P < .05 comparing the inhibitors used. (C) Human SK-N-MC cells were treated for 24 hours with the indicated agents and then cell lysates (30 μg) were measured for caspase-3 activation using a fluorogenic substrate. Representative data are shown from 2 independent experiments, each performed in triplicate, as the mean plus or minus SD. (D) Human umbilical vein endothelial cells (HUVECs) were plated until they were 70% to 80% confluent, and then treated with increasing concentrations of IPSI (μM) or bortezomib (nM) for 24 hours, followed by evaluation by WST-1 for cell viability in triplicate. The data shown are the mean plus or minus SD from experiments performed in triplicate. (E) Human peripheral blood mononuclear cells (PBMCs) were plated and exposed to IPSI-001 and bortezomib. Viable cells were assayed 24 hours later using WST-1 reagent. *P < .05 for IPSI-001 treatment compared with bortezomib.