JS-K induces apoptosis in MM cells associated with the extrinsic and intrinsic apoptotic pathways. (A) MM.1S (□), RPMI-8226 (■), and OPM1 (□) cells were treated with JS-K at their IC50 values (0.6 μM, 1.2 μM, and 0.3 μM, respectively) for 0 to 48 hours, and apoptosis was then assessed by flow cytometry following Annexin V staining. Data represent means (± SD) of triplicate experiments. (B) Cleavage of PARP and the initiator caspase-8 and caspase-9 were determined by Western blotting of MM.1S cells treated with 0.6 μM JS-K for 0 to 36 hours (left panel), or with 0 to 1.2 μM JS-K for 24 hours (right panel). (C) MM.1S cells were treated with JS-K (0–2.5 μM) for 48 hours, with (■) or without (□) pretreatment by z-VAD-fmk (100 μM). Cell death was assessed by flow cytometry after PI staining. Data represent means (± SD) of triplicate experiments. *P < .01, as compared with no z-VAD-fmk pretreatment. (D) MM.1S cells were treated with 0.6 μM JS-K for 0 to 24 hours, and cell-surface CD95/Fas expression was then assessed by flow cytometry. Fold increase of mean surface CD95/Fas expression induced by JS-K is plotted relative to untreated control (left panel). Data represent means ± SD of triplicate experiments. *P < .01. The histogram plot shows the percentage of cells positive for cell-surface CD95/Fas at 0 hours and 24 hours after JS-K exposure (right panel). (E-F) MM.1S cells were treated with JS-K (0-1.2 μM) for 24 hours, followed by immunoblotting for Bcl-2 family proteins (E) or release of mitochondrial proteins into the cytosol (F).
