Article Figures & Data
Figures
- Figure 1.
Sorafenib inhibits cell proliferation and induces apoptosis in HCC cells. A, inhibition of cell proliferation. Sorafenib was added to PLC/PRF/5 and HepG2 cells and cultured for 72 hours in RPMI 1640 containing 10% FBS. The experiment was done at least twice (○, experiment 1; •, experiment 2). B, induction of DNA fragmentation detected by ELISA. Sorafenib was added to PLC/PRF/5 and HepG2 cells and cultured for 48 hours in complete culture medium in the presence of 10% FBS (•) or 0.1% BSA (○). OD, absorbance. C, TUNEL staining. Cells were treated with sorafenib in the presence of complete culture medium containing 10% FBS. ZVAD-fmk was added 2 hours before sorafenib treatment for 48 hours. Percentage of TUNEL-positive cells was quantified using Cellomic ArrayScan II image analysis system at ×40 magnification. D, Hoechst staining. Specific apoptotic cells, including both nuclear condensation and/or nuclear fragmentation phenotypes, were visualized and quantified using Cellomic ArrayScan II image analysis system. Assays were done in triplicate. Columns, mean (n = 3) of three independent determinations; bars, SE. *, P < 0.05, one-way ANOVA when compared with DMSO controls. E, cell cycle distribution. Cells were treated with sorafenib in the presence of complete culture medium containing 10% FBS for 24 hours and stained with propidium iodide. Cell cycle distribution was assessed using flow cytometry and quantified by Modfit software. The percentage of cells in G1, S, or G2-M phase was calculated after gating sub-G0 population. Each value represents the percentage of cells in the noted cell cycle phases and is the average of two independent determinations. All results are representative of two separate experiments.
- Figure 2.
Sorafenib inhibits RAF/MEK/ERK signaling in PLC/PRF/5 (A) and HepG2 (B) cells. Cells were treated with compounds in RPMI 1640 with 0.1% BSA for 2 hours or followed by addition of hepatocyte growth factor (HGF; 25 ng/mL) for 10 minutes. Cells were lysed and 20 μg of soluble protein was separated by electrophoresis on a SDS-PAGE gel. Protein phosphorylation was detected by Western blot analysis.
- Figure 3.
Sorafenib down-regulates cyclin D1 in HCC cells. PLC/PRF/5 (A) and HepG2 (B) cells were treated with compound for 5 or 16 hours in RPMI 1640 containing 10% FBS. After treatment, cells were lysed and 20 μg of soluble protein were separated by electrophoresis on a SDS-PAGE gel. Protein levels were detected by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin was used as a loading control.
- Figure 4.
Sorafenib reduces phosphorylation of eIF4E and down-regulates Mcl-1 levels in HCC cells, independent of MEK/ERK. PLC/PRF/5 (A) and HepG2 (B) cells were treated with compound for 2 or 16 hours in RPMI 1640 with 10% FBS. After treatment, cells were lysed and 20 μg of soluble protein were separated by electrophoresis on a SDS-PAGE gel. Protein levels were detected by Western blot analysis. GAPDH or eIF4E was used as a loading control.
- Figure 5.
Sorafenib produces robust efficacy against PLC/PRF/5 HCC tumors in mice. PLC/PRF/5 tumors cells (5 × 106 per animal) were implanted s.c. in the flank of athymic mice as described in Materials and Methods in two independent experiments. Treatment was initiated on day 11 or 12 when all groups had tumors averaging 140 to 160 mg in size. Sorafenib tosylate was administered p.o. at 10, 30, or 100 mg/kg, qd × 16 days or qd × 21 days. There was no lethality in any group. Daily p.o. administration of sorafenib tosylate produced dose-dependent TGI and at 100 mg/kg; partial tumor regressions were observed in 5 of 10 animals. Points, mean tumor weight (n = 10); bars, SE. *, P < 0.001.
- Figure 6.
In vivo mechanism of action of sorafenib in PLC/PRF/5 HCC tumors. Sorafenib tosylate was administered once daily for 5 days at the indicated dose levels to female SCID mice with tumors measuring 200 to 300 mg in size. Tumors were collected 3 hours after the last treatment. Tumors were either homogenized for analysis by Western blot or formalin fixed for 24 hours and then analyzed by immunohistochemistry (IHC). A, sorafenib reduces the microvessel density, as measured by CD34 staining; the amount of phosphorylation of ERK, as measured by phosphospecific antibodies to phospho-ERK (p-ERK); and induces apoptosis, as measured by the increase in staining of TUNEL-positive area in PLC/PRF/5 tumors in mice. B, sorafenib reduces the phosphorylation level of eIF4E in PLC/PRF/5 HCC tumors in mice. C, quantification of microvessel (CD34 staining) and TUNEL-positive areas from immunohistochemical analysis of PLC/PRF/5 tumors. Columns, mean (five samples per group); bars, SE. *, P < 0.05, treated group versus vehicle group by one-way ANOVA Dunnet's multiple comparison test.
Tables
- Table 1.
Effect of sorafenib tosylate against s.c. PLC/PRF/5 human HCC xenografts
Sorafenib tosylate (mg/kg/dose) * Experiment 1 Experiment 2 Percent TGI [(1 − T/C) × 100], qd × 21 Maximum percent net body weight loss Percent TGI [(1 − T/C) × 100], qd × 16 Maximum percent net body weight loss Control −2 17 0 6 Vehicle 0 17 −7 8 10 49 † 16 − — 30 78 †, ‡ 12 75 † 12 100 — — 82 † (5/10 PR) 16 -
↵* Sorafenib tosylate was administered p.o. once daily for 16 or 21 days at dose levels of 10, 30, and 100 mg/kg when all animals in the study had established tumors averaging from 140 to 160 mg.
-
↵† P < 0.001, significantly different from the control group.
-
↵‡ P < 0.001, significantly different from the 10 mg/kg dose level.
-
Volume 66, Issue 24
