Combination of low doses of NPI-0052 and bortezomib induces synergistic MM cell death. MM.1S (A), U266 (B), RPMI-8226 (C), INA-6 (D), OPM-2 (E), Dox-40 (F), or MM.1R (G) cells were treated with NPI-0052, bortezomib, or combined NPI-0052 plus bortezomib and assessed for viability using MTT assays. The concentrations of drugs, either alone or in combination, were as follows: MM.1S cells: 1 nM NPI-0052, 3 nM bortezomib, or NPI-0052 (1 nM) plus bortezomib (3 nM); U266 cells: 7 nM NPI-0052, 1 nM bortezomib, or NPI-0052 (7 nM) plus bortezomib (1 nM); RPMI-8226 cells: 10 nM NPI-0052, 10 nM bortezomib, or NPI-0052 (10 nM) plus bortezomib (10 nM); INA-6 cells: 5 nM NPI-0052, 5 nM bortezomib, or NPI-0052 (5 nM) plus bortezomib (5 nM); OPM-2 cells: 5 nM NPI-0052, 7 nM bortezomib, or NPI-0052 (5 nM) plus bortezomib (7 nM); Dox-40 cells: 5 nM NPI-0052, 10 nM bortezomib, or NPI-0052 (5 nM) plus bortezomib (10 nM); and MM.1R cells: 5 nM NPI-0052, 3 nM bortezomib, or NPI-0052 (5 nM) plus bortezomib (3 nM). Data presented are means plus or minus SD (n = 3; P < .005 for all cell lines). A CI of less than 1 indicates synergy. (H) MM.1S cells were treated with the indicated concentrations of NPI-0052, bortezomib, or combined NPI-0052 plus bortezomib for 12 hours and assessed for apoptosis using annexin V/PI staining assays by flow cytometry. Percentage of apoptotic cells (annexin V⁺/PI⁻) were as follows: control cells, 3.5%; NPI-0052–treated cells, 3.6%; bortezomib-treated cells, 5%; and NPI-0052 plus bortezomib–treated cells, 25%. Data presented are means plus or minus SD (n = 2; P < .04). (I) MM.1S cells were treated with the indicated concentrations of NPI-0052, bortezomib, or combined NPI-0052 plus bortezomib for 12 hours and assessed for cell death using a live and dead cell assay using fluorescent microscopy (Leica Microsystems AF600, Bannockburn, IL); (live cells are green; dead cells are red). Data presented are means plus or minus SD (n = 3; P < .05).