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. Author manuscript; available in PMC: 2010 Jul 21.

Published in final edited form as: Oncogene. 2009 Nov 23;29(8):1190–1202. doi: 10.1038/onc.2009.403

Sorafenib induces apoptosis in MM1.S myeloma cell line and myeloma patient cells. Time-dependent increase in the apoptotic cells was observed when MM1.S multiple myeloma (MM) cells were treated with increasing doses of sorafenib (a). Annexin V-fluorescein isothiocyanate (FITC) staining is represented on the X axis and propidium iodide (PI) staining is represented on the Y axis. The proportion of cells in each quadrant is as indicated in the figure. There is a time-dependent decrease in viable cells (lower left quadrant) with concomitant increase in apoptotic (lower right quadrant) and necrotic (upper right quadrant) cells. When MM1.S MM cells were treated with sorafenib (5 μm), induction of apoptosis in MM cells was accompanied by a time-dependent (0, 3 and 6 h) cleavage of caspase 3, caspase 8 and caspase 9 as shown by flow cytometry (b). Induction of apoptosis is also confirmed by the timedependent (0, 1, 2, 4 and 6) cleavage of caspases 3, 8 and 9 as well as PARP as shown by immunoblotting (b). Sorafenib induces apoptosis of freshly isolated patient MM cells when cultured with the indicated drug concentrations for 36–48 h as measured using Apo 2.7-PE staining and flow cytometry. Sorafenib concentrations (μm) are indicated and percentage of apoptotic cells (percentage of cells expressing membrane Apo 2.7) is indicated on the Y axis (c). Results from 10 different patients are shown; proportion of cells positive for Apo 2.7 is shown on the Y axis. In addition, whole marrow mononuclear cells were treated with 5 or 10 μm sorafenib for 48 h and the CD38/CD45 plasma cell compartment was examined using flow cytometry (d). CD38 expression is denoted on the Y axis and CD45 expression on the X axis. Plasma cells were identified as those cells staining brightly for CD38. Both the CD45 positive and negative MM cells appear to be sensitive to the cytotoxic effects of sorafenib. Primary patient cells were treated with indicated concentrations of sorafenib for 48 h and cytotoxic effects of sorafenib on patient cells measured using MTT assays (e). In the above experiments it must be noted that all groups (control cells and sorafenib treated cells) were analysed at the same time. Control groups were left untreated for the duration of the experiment and analysed along with the sorafenib-treated cells. In the case of drug-treated cells, sorafenib was added at different time points to cells in culture that were set up at the same time. The duration of incubation with sorafenib is indicated in respective figures.

Sorafenib induces apoptosis in MM1.S myeloma cell line and myeloma patient cells. Time-dependent increase in the apoptotic cells was observed when MM1.S multiple myeloma (MM) cells were treated with increasing doses of sorafenib (a). Annexin V-fluorescein isothiocyanate (FITC) staining is represented on the X axis and propidium iodide (PI) staining is represented on the Y axis. The proportion of cells in each quadrant is as indicated in the figure. There is a time-dependent decrease in viable cells (lower left quadrant) with concomitant increase in apoptotic (lower right quadrant) and necrotic (upper right quadrant) cells. When MM1.S MM cells were treated with sorafenib (5 μm), induction of apoptosis in MM cells was accompanied by a time-dependent (0, 3 and 6 h) cleavage of caspase 3, caspase 8 and caspase 9 as shown by flow cytometry (b). Induction of apoptosis is also confirmed by the timedependent (0, 1, 2, 4 and 6) cleavage of caspases 3, 8 and 9 as well as PARP as shown by immunoblotting (b). Sorafenib induces apoptosis of freshly isolated patient MM cells when cultured with the indicated drug concentrations for 36–48 h as measured using Apo 2.7-PE staining and flow cytometry. Sorafenib concentrations (μm) are indicated and percentage of apoptotic cells (percentage of cells expressing membrane Apo 2.7) is indicated on the Y axis (c). Results from 10 different patients are shown; proportion of cells positive for Apo 2.7 is shown on the Y axis. In addition, whole marrow mononuclear cells were treated with 5 or 10 μm sorafenib for 48 h and the CD38/CD45 plasma cell compartment was examined using flow cytometry (d). CD38 expression is denoted on the Y axis and CD45 expression on the X axis. Plasma cells were identified as those cells staining brightly for CD38. Both the CD45 positive and negative MM cells appear to be sensitive to the cytotoxic effects of sorafenib. Primary patient cells were treated with indicated concentrations of sorafenib for 48 h and cytotoxic effects of sorafenib on patient cells measured using MTT assays (e). In the above experiments it must be noted that all groups (control cells and sorafenib treated cells) were analysed at the same time. Control groups were left untreated for the duration of the experiment and analysed along with the sorafenib-treated cells. In the case of drug-treated cells, sorafenib was added at different time points to cells in culture that were set up at the same time. The duration of incubation with sorafenib is indicated in respective figures.