By identifying the mechanism of therapeutic effects of the combination of interferon alpha (IFNα)-2b and 5-fluorouracil (5-FU) on advanced hepatocellular carcinoma (HCC) with portal venous invasion1, we recently found that 5-FU increased both the expression and secretion of transforming growth factor (TGF)-β in HepG2 cells. In this study, we analyzed the effects of 5-FU on TGF-β-related signaling molecules.
Hepatoma cells (HepG2 and HuH7) were treated with 5-FU, IFNα-2b, and the combination of 5-FU and IFNα-2b. The total and/or phosphorylated protein levels of TGF-β-related signaling molecules were detected by western blot analysis. Additionally, the effects of above-mentioned treatments on the epithelial–mesenchymal transition (EMT) of the cells were evaluated by performing invasion and migration assays.
With respect to the TGF-β-induced apoptosis signals, 5-FU inhibited not only the phosphorylation of SMAD2, but also reduced the total protein levels of SMAD2, SMAD4, and pINK4b. Conversely, 5-FU stimulated the phosphorylation of TGF-β-induced EMT molecules, such as ERK and JNK, but not p38MAPK. Accordingly, the protein levels of E-cadherin were reduced in the cells treated with 5-FU. On the other hand, IFNα-2b did not affect the levels of TGF-β-induced EMT molecules, whereas the combination of 5-FU and IFNα-2b neutralized the effects of 5-FU on TGF-β-related signaling molecules and restored their protein levels to those observed in the control. Interestingly, the phosphorylated protein levels of SMAD2 and the total protein levels of E-cadherin and p15INK4b increased in 5-FU-stimulated HUH7 cells, but not in HepG2 cells. Furthermore, 5-FU stimulated both cell invasion and migration in HepG2 cells, whereas the combination of 5-FU and IFNα-2b abolished these effects of 5-FU.
Our data suggest that 5-FU induces the EMT of hepatoma cells through TGF-β, and that the higher efficacy of the combination therapy of 5-FU and IFNα-2b results from the inhibition of these effects of TGF-β. The differences observed between HepG2 and HUH7 cells in response to the stimulation with 5-FU indicate that the efficacy of the therapy may differ between patients with hepatitis B (HBV) or C virus (HCV) background.
Clinical trial identification
Legal entity responsible for the study
Iwate Medical University
Japan Society for the Promotion of Science (JSPS)
All authors have declared no conflicts of interest.