Figures & data
Figure 1. Sorafenib inhibits FLT3 signaling in 32D cells expressing FLT3-ITD but paradoxically induces Erk activity and proliferation in 32D control cells. (A) Immunoblot analyses of Erk activity in 32D-FLT3-ITD (left panel) and 32D (right panel) cell lines. 32D and 32D-FLT3-ITD cells were incubated with different concentrations of sorafenib for 48 h. Cells with DMSO served as control. Whole cell extracts (20 μg) were analyzed by immunoblotting using an antibody against phosphorylated Erk (phospho Erk, 42 and 44 kDa). Total Erk1/2 and β-actin served as loading controls. (B) Flow cytometric analysis of BrdU-Alexa Fluor 488/propidium iodide (PI)-stained cells. 32D cells were treated or untreated with sorafenib for 48 h. (C) Cell cycle determination of five independent experiments using the murine 32D cell line. Shown are cells in G1- and S-phase. Data are presented as percent of control without sorafenib (left). The plot is a “min–max” plot and shows an increase of cells in S-phase depending on increasing sorafenib concentration (p = 0.0012).
Figure 2. Sorafenib enhances glycolytic and respiratory activity in 32D but leads to decreased glycolysis and respiration in 32D-FLT3-ITD cells. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) decline in 32D-FLT3-ITD cells but increase significantly in 32D cells after exposure to sorafenib for 24 h (p < 0.0001 ECAR; p < 0.0002 OCR). Addition of U0126 (10 μM) abrogates this effect in 32D cells. ECAR was determined after the addition of glucose, OCR was measured in basal medium without glucose.
Figure 3. Sorafenib inhibits FLT3 signaling at low concentration and induces apoptosis in FLT3-ITD expressing cells but not in AML cells lacking FLT3-ITD. (A) Cell proliferation analyzed by CellTiter 96® AQueous One Solution Cell Proliferation Assay using 72 h sorafenib treated or untreated MV4-11 and U937 cells. Data were recorded as optical densities (ODs) (measured from six replicate wells). Standard deviation (SD) is indicated (bars). MV4-11 and U937 cells were treated or untreated with sorafenib (200 nM) for 72 h (B,C). (B) Annexin V-FITC/propidium iodide (PI)-stained cells were analyzed by flow cytometry. Upper right quadrants: apoptotic cells. (C) Immunoblot analyses of Bad phosphorylation. Whole-cell extracts were analyzed by Mini-Protean TGX stain-free gel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control.
Figure 4. Sorafenib inhibits MAPK signaling in MV4-11 but activates it in U937 cells. Immunoblot analysis of phosphorylated C-Raf and Erk using 48 h sorafenib (50, 100, 200 nM) treated or untreated MV4-11 (A) and 72 h sorafenib (200 nM) treated or untreated U937 cells (B). Total C-Raf, Erk1/2 and GAPDH served as loading controls. (C) Flow cytometric analysis of BrdU-Alexa Fluor 488/propidium iodide (PI)-stained cells. U937 cells were treated or untreated with sorafenib for 48 h. The percentage of cells in S-phase as well as G1/G2 is displayed.
Figure 5. Mek1/2 inhibitor blocks sorafenib-induced Erk2 activity in U937 cells. Immunoblot analysis of p70S6 kinase and Erk1/2 phosphorylation. U937 cells were treated with Mek1/2 inhibitor U0126 (10 μM) and/or sorafenib (200 nM) for 72 h. MV4-11 cells were treated with 200 nM sorafenib for 72 h. Untreated cells were used as controls. Total Erk1/2, p70S6 kinase α and GAPDH served as loading controls.
Figure 6. Sorafenib induces paradoxical effect in primary AML cells lacking FLT3-ITD and untransformed blood cells. (A) Cells from a leukemic patient suffering from AML lacking FLT3-ITD were isolated, frozen and thawed. After thawing, cells were incubated in medium supplemented with 20% fetal calf serum and incubated for 24 h. Cells were then washed, lysed and immunoblot was performed as described in the “Materials and methods” section. A slight increase of phosphorylated Erk and Erk can be seen (representative experiment). MV4-11 cells with FLT3-ITD treated for 24 h with sorafenib (50 nM) served as positive control. (B) Immunoblot analysis of Erk activity in peripheral blood mononuclear cells from a patient suffering from thyroid carcinoma (TC) with or without (4 weeks after) sorafenib therapy and a healthy donor (control). Whole cell extracts (20 μg) were analyzed by immunoblotting using an antibody against phosphorylated Erk. Total Erk1/2 and β-actin served as loading controls.
