CEP-18770 induces apoptosis in human MM cell lines and in purified primary human MM explant cultures. (A) RPMI-8226, U266, and K562 cells were treated with CEP-18770 (20 nM) or BOR (10 nM) for the indicated times. Levels of cleaved fragments of caspase 3, caspase 7, and caspase 9 (CF) and of full-length (115 kDa) and cleaved fragment of PARP (85 kDa) were determined by immunoblotting on whole-cell lysates with the indicated antibodies; α-tubulin protein expression was included as loading control. (B) Apoptosis was measured by flow cytometry with the mitochondrion-permeable dye TMRM in RPMI-8226, U266, and K562 cells after 12 and 24 hours of CEP-18770 (20 nM) or BOR (10 nM) treatment. Histograms are representative of the percentage of apoptotic cells in one of 3 experiments (C) Human MM cell lines were treated with control diluent or CEP-18770 (20 nM) for 24 hours, percentages of apoptosis were measured after TMRM staining by flow cytometry. Histograms are representative of the percentage of apoptotic cells in one of 3 experiments (D) CD138⁺ PC, obtained from 2 patients with MM, were separated by positive selection and PC were seeded on a BMSC monolayer and treated with CEP-18770 or inactive deboronated CEP-18770 for 72 hours at different concentrations. The percentage of purified MM cells undergoing apoptosis as measured by staining with TMRM and flow cytometry is indicated. (E) CD138⁺ PC obtained from 8 patients with MM (PC > 10%) as described above, were treated with CEP-18870 (20 nM) or bortezomib (BOR) (10 nM) for 72 hours. Apoptosis was measured by flow cytometry after TMRM staining. Histograms represent the percentage of viable cells normalized versus control samples.