GCS-100 inhibits proliferation, induces apoptosis, and modifies cell-cycle profile of myeloma cell lines. (A) Alamar Blue assessment of the effect of GCS-100 on proliferation of RPMI 8226 (■), U266 (●), and OPM-2 (▴) cells. Cells were cultured with GCS-100 (0-1500 μg/mL) for 48 hours and proliferation assessed compared with nonexposed control cells. Results are the mean of 3 independent experiments ± SEM. *Significant difference (P ≤ .01) compared with untreated cells. (B) Apoptosis induction of RPMI 8226 (● indicates RPMI 8226; and ▾ indicates U266) cells by GCS-100 was assessed by flow cytometry. Cells were cultured with GCS-100 (0-800 μg/mL) for 72 hours and then examined for binding of annexin V–FITC and uptake of DAPI. Annexin V–positive/DAPI-negative cells (● indicates RPMI 8226; and ▾ indicates U266) were classified as early apoptotic, whereas annexin V–positive cells (● indicates RPMI 8226; and ▾ indicates U266) were classified as apoptotic/necrotic. The results of 3 independent experiments ± SEM are shown. *Significant difference (P ≤ .01) compared with untreated cells. (C-D) Cell-cycle changes effected by GCS-100. RPMI 8226 (C) and U266 (D) cells were cultured with GCS-100 (0-800 μg/mL) for 48 hours. The cells were then permeabilized and stained with propidium iodide (PI). Cell-cycle profile was assessed by flow cytometry, and the proportion of cells in the sub-G₁ fraction, G₁ phase, S phase, and G₂/M phase was measured. Representative histograms of cells treated with 0, 250, and 800 μg/mL GCS-100 are shown (concentration is displayed in the top right of histogram).