Sorafenib inhibits proliferation and invasion of human hepatocellular carcinoma cells via up-regulation of p53 and suppressing FoxM1

Abstract

Aim: Forkhead box M1 (FoxM1) is a transcription factor that plays important roles in the pathogenesis and progression of human cancers, including hepatocellular carcinoma (HCC). The aim of this study was to examine the involvement of FoxM1 in the anti-cancer action of sorafenib, a multikinase inhibitor, in human HCC cells.

Methods: HCC cell lines HepG2 and HuH-7 were tested. Cell viability was examined using MTT assay and cell invasion was determined with Transwell migration assay. The relevant mRNA expression was determined with RT-PCR, and the proteins were detected using Western blotting and immunofluorescence assays. RNA interference was used to modify the expression of p53 and FoxM1. HuH-7 cell line xenograft mice were used for in vivo study, which were treated with sorafenib (40 mg/kg, po) daily for 3 weeks.

Results: Sorafenib (2-20 μmol/L) inhibited the proliferation of the cells in dose- and time-dependent manners with an IC50 value of nearly 6 μmol/L at 48 h. Sorafenib (6 μmol/L) markedly suppressed the cell invasion. Furthermore, sorafenib (2-6 μmol/L) dose-dependently decreased the expression of FoxM1, MMP-2, and Ki-67, and up-regulated that of p53 in the cells. Silencing p53 abolished the decrease of FoxM1 and increase of p53 in sorafenib-treated cells. Silencing FoxM1 significantly reduced the expression of MMP-2 and Ki-67, and enhanced the anti-proliferation action of sorafenib in the cells, whereas overexpression of FoxM1 increased the expression of MMP-2 and Ki-67, and abrogated the anti-proliferation action of sorafenib. In the xenograft mice, sorafenib administration decreased the tumor growth by 40%, and markedly increased the expression of p53, and decreased the expression of FoxM1, MMP-2, and Ki-67 in tumor tissues.

Conclusion: Sorafenib inhibits HCC proliferation and invasion by inhibiting MMP-2 and Ki-67 expression due to up-regulation of P53 and suppressing FoxM1.

Figure 1

Sorafenib significantly inhibits the proliferation and invasion of HepG2 and HuH-7. The cell lines HepG2 and HuH-7 were exposed to sorafenib at increasing concentrations at 48 h. The IC₅₀ concentration was approximately 6 μmol/L (A). Cell proliferation was mostly inhibited at 48 h when both cell lines were exposed to sorafenib at 6 μmol/L (B). To eliminate the influence of cell death on the experiment, we included the control group without sorafenib treatment. The cell viability of the control group was set at 100% and was compared with that of other groups. The working concentration of sorafenib is 6 μmol/L. The effect of sorafenib on cell invasion was tested by the Transwell assay (C). The number of positive cells is presented in (D). To each well of 24-well microtiter culture plates was added 500 μL of medium that was used for cell invasion assay, and the volume of the 96-well microtiter culture plates used for the MTT assay was 100 μL per well. The experiment shown is representative of at least three separate experiments. Statistical significance was defined as ^bP<0.05.

Figure 2

FoxM1 expression is inhibited by sorafenib in a time-dependent manner. FoxM1 mRNA levels in sorafenib-treated cells were evaluated by real-time PCR. Significant changes were observed in the FoxM1 mRNA levels after sorafenib treatment (6 μmol/L) in hepatoma cells at 48 h (A). The reduced FoxM1 expression following sorafenib treatment was observed by Western blotting and was time dependent (B). Equal sample loading was verified by stripping the blots and re-probing with anti β-actin antibody. The band density of each protein was normalized with β-actin and expressed as the mean±SD compared with the controls from 3 independent experiments. HuH-7 cells were treated with 6 μmol/L sorafenib for 48 h. Immunofluorescence was conducted as described in the Materials and Methods for FoxM1 (green). Representative pictures are shown (C) with a magnification of 200×. The experiment shown is representative of at least three separate experiments. At least six randomly selected fields were examined in each of three separate experiments. Statistical significance was defined as ^bP<0.05.

Figure 3

Sorafenib significantly inhibits the expression of MMP2 and Ki-67 by down-regulating FoxM1. FoxM1, MMP2, and Ki-67 levels as measured by real-time RT-PCR (A) and Western blotting (B) in two hepatoma cells. FoxM1 expression is inhibited by sorafenib in a concentration-dependent manner. Equal sample loading was verified by stripping the blots and re-probing with the anti β-actin antibody. The band density of each protein was normalized with β-actin and expressed as the mean±SD compared with the controls from 3 independent experiments. The experiment shown is representative of at least three separate experiments. Statistical significance was defined as ^bP<0.05.

Figure 4

Regulation of p53 expression by siRNA, followed by sorafenib treatment. p53 down-regulation by siRNA can enhance FoxM1 expression in hepatoma cells (A). Down-regulation of p53 by siRNA, followed by sorafenib treatment as measured by Western blotting. p53 and FoxM1 expression was determined (B). Equal sample loading was verified by stripping the blots and re-probing with the anti β-actin antibody. The band density of each protein was normalized with β-actin and expressed as the mean±SD compared with the controls from 3 independent experiments. The experiment shown is representative of at least three separate experiments. Statistical significance was defined as ^bP<0.05.

Figure 5

Effect of FoxM1 on cell invasion and proliferation factors. MMP-2 and Ki-67 expression as measured by Western blotting in two hepatoma cells transfected with shRNA or cDNA. MMP-2 and Ki-67 expression was significantly inhibited when the two cell lines were transfected with FoxM1 shRNA (A). MMP-2 and Ki-67 expression was enhanced when transfected with FoxM1 cDNA (B). No notable differences were observed in p53 expression. The band density of each protein was normalized with β-actin and expressed as the mean±SD compared with the controls from 3 independent experiments. The experiment shown is representative of at least three separate experiments. Statistical significance was defined as ^bP<0.05.

Figure 6

Regulation of FoxM1 expression by shRNA or cDNA, followed by sorafenib treatment. The growth viability of HCC cells was decreased by sorafenib treatment and shRNA transfection. Surprisingly, the proliferation of the two cell lines was inhibited by as much as 70% when sorafenib treatment was combined with shRNA transfection. Significant differences were observed compared with the no-treatment group. Down-regulation of FoxM1 expression by shRNA promoted sorafenib-induced cell growth inhibition in hepatoma cells (A and B). No obvious differences were observed when sorafenib treatment was combined with cDNA transfection. Overexpression of FoxM1 by FoxM1 cDNA transfection abrogated the sorafenib-induced cell growth inhibition in hepatoma cells (C and D). The experiment shown is representative of at least three separate experiments. Statistical significance was defined as ^bP<0.05.

Figure 7

FoxM1 mediates the anti-tumor effect of sorafenib in a xenograft model. Effect of FoxM1 expression on tumor growth (A). Sorafenib inhibited tumor growth (B). Knocked-down FoxM1 expression in combination with sorafenib treatment strongly inhibited tumor size (C). The reduction in tumor size was not significant when FoxM1 expression was up-regulated in combination with sorafenib treatment (D). p53, FoxM1, MMP-2, and Ki-67 expression was determined via immunohistochemical analysis in tissues with and without sorafenib treatment. The number of positive cells was also quantified (E). Statistical significance was defined as ^bP<0.05.