IPI-504 disrupts BiP/Hsp90 complexes, inhibits UPR activation, and synergistically induces apoptosis with bortezomib in resistant cells. (A) Jeko-1, JBR, Z-138, and ZBR cells were incubated for 72 hours with increasing concentrations of 17-AAG or IPI-504 and cytotoxicity was assessed by the 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide assay. The relative number of proliferating cells compared with control, untreated cells are presented as the mean ± SD of triplicate assays. (B) Relationship between sensitivity/resistance to bortezomib (bz) status and efficacy of IPI-504–bz combination in 20 MCL samples. Cells were pretreated for 24 hours with 0.01, 0.05, 0.1, or 0.5μM IPI-504, followed by an additional 16-hour exposure to 2.5, 5, 10, or 20nM bortezomib. Cytotoxicity was determined by Annexin V staining, and the CIs were calculated in both bortezomib-resistant (bz LC₅₀ > 20nM) and bortezomib-sensitive (bz LC₅₀ < 20nM) groups. Shown are the cytotoxicity and the mean CI values calculated on cell treatment with 10nM bortezomib and 0.5μM IPI-504. (C-D) JBR cells and cells from a representative bortezomib-resistant MCL sample were treated as above with 0.5μM IPI-504 and 20nM bortezomib. The levels of UPR proteins [BiP/Grp78, phospho–elongation initiation factor 2α (eIF2α), spliced (s) and unspliced (u) XBP-1, CHOP] and apoptosis hallmark proteins [NOXA, poly(ADP-ribose) polymerase (PARP) cleavage] were determined by Western blot. Mitochondrial transmembrane potential loss (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 cleavage were evaluated by flow cytometry. Percentages inside each chart refer to the cell population positive for the corresponding label.