Combination of interferon-alpha and 5-fluorouracil inhibits endothelial cell growth directly and by regulation of angiogenic factors released by tumor cells

Abstract

Background: The combination therapy of interferon (IFN)-alpha and 5-fluorouracil (5-FU) improved the prognosis of the patients with hepatocellular carcinoma (HCC). To determine the molecular mechanisms of the anti-tumor and anti-angiogenic effects, we examined the direct anti-proliferative effects on human umbilical vein endothelial cells (HUVEC) and indirect effects by regulating secretion of angiogenic factors from HCC cells.

Methods: The direct effects on HUVEC were examined by TUNEL, Annexin-V assays and cell cycles analysis. For analysis of the indirect effects, the apoptosis induced by the conditioned medium from HCC cell treated by IFN-alpha/5-FU and expression of angiogenic factors was examined.

Results: IFN-alpha and 5-FU alone had anti-proliferative properties on HUVEC and their combination significantly inhibited the growth (compared with control, 5-FU or IFN alone). TUNEL and Annexin-V assays showed no apoptosis. Cell cycle analysis revealed that IFN-alpha and 5-FU delayed cell cycle progression in HUVEC with S-phase accumulation. The conditioned medium from HuH-7 cells after treatment with IFN/5-FU significantly inhibited HUVEC growth and induced apoptosis, and contained high levels of angiopoietin (Ang)-1 and low levels of vascular endothelial growth factor (VEGF) and Ang-2. Knockdown of Ang-1 in HuH-7 cells abrogated the anti-proliferative effects on HUVEC while knockdown of Ang-2 partially rescue the cells.

Conclusion: These results suggested that IFN-alpha and 5-FU had direct growth inhibitory effects on endothelial cells, as well as anti-angiogenic effects through regulation of angiogenic factors released from HCC cells. Modulation of VEGF and Angs secretion by IFN-alpha and 5-FU may contribute to their anti-angiogenic and anti-tumor effects on HCC.

Figure 1

MTT growth inhibitory assay. 5-FU alone inhibited HUVEC cells growth (A). IFN-α alone slightly inhibited HUVEC cell growth, even when used at a high concentration (10,000 units/ml) (B). Significant synergistic effects for IFN-α and 5-FU were observed at 0.05 μg/ml of 5-FU and 500 or 5,000 units/ml of IFN-α (p < 0.05), but not at 0.5 or 5 μg/ml of 5-FU plus 500 units/ml of IFN-α (C). A significant difference was observed in cell numbers on day 7 between the IFN/5-FU combination group and the other groups (D).

Figure 2

Flow cytometric analysis of cell cycle progression in HUVEC cells treated with or without IFN-α (500 units/ml) and/or 5-FU (0.5 μg/ml). To synchronize the cell cycle in G0-G1, HUVEC cells were first pre-treated with 2 μM aphidicolin for 16 h. Cells were collected 12, 24, 48 and 72 h later. After pre-treatment by aphidicolin, the majority of cells (86.3%) were in G0-G1. At 24 h, IFN-α alone and IFN/5-FU increased the number of cells with S-phase DNA content. At 48 h and 72 h, IFN/5-FU still resulted in S-phase accumulation.

Figure 3

BrdUrd labeling index and tube formation in vitro. (A) In vitro angiogenesis assay showed that HUVEC formed vessel-like structures (tubes) when plated on Matrigel-coated wells. 5-FU treatment did not inhibit tube and network formation. In contrast, IFN-α caused thinner or only weakly-stained tube-like structures. IFN/5-FU also inhibited tube formation compared to the control and caused only weak staining of the tube-like structures, similar to IFN-α alone. (B) 5-FU alone and IFN/5-FU caused significant decreases in BrdUrd labeling index compared with the control and IFN-α alone (p < 0.01). There was no difference in the index between 5-FU alone and IFN/5-FU combination (A, B). There was a significant difference in the number of capillary connections, defined as cross-points consisting of three tubes, among the control, 5-FU alone and IFN-α, IFN/5-FU (p < 0.01).

Figure 4

Effect of 5-FU alone and IFN-α, IFN/5-FU on apoptosis of HUVEC. (A) TUNEL assay showed limited number of TUNEL-positive cells in each treatment. (B) IFN/5-FU did not induce apoptosis of HUVEC in annexin-V assay. The percentage of Annexin-V positive cells is shown in figures. (C) Serum-free CM from control culture of HuH-7 significantly promoted HUVEC growth. Supernatants from HuH-7 treated by IFN-α (500 units/ml) and 5-FU (0.5 μg/ml) (CM-IFN/5-FU) significantly inhibited the growth of HUVEC. (D) Growth inhibition of CM-IFN/5-FU was due to induction of apoptosis. The number of TUNEL-positive cells in CM-IFN/5-FU was significantly higher than in other conditioned media (Figure 4D).

Figure 5

Concentrations of angiogenic factors (VEGF, Ang-1 and Ang-2) in the supernatants of HCC cells treated without (control) or with IFN-α, 5-FU or their combination, measured by ELISA assay kits.

Figure 6

(A) Knock-down of Ang-1 or Ang-2 efficiently represses the expression of Ang-1 or Ang-2 mRNA in HuH-7 cells. HuH-7 cells were transfected to Ang-1 or Ang-2 siRNA. Forty eight hours after the transfection, we evaluated the expression of Ang-1 or Ang-2 mRNA by real time RT-PCR. Values shown are relative induction of the indicated genes. (B) The supernatant of HuH-7 cells after knockdown of Ang-1 completely abrogated the anti-proliferative effects of the conditioned medium from IFN/5-FU treated HuH-7 cells. HuH-7 cells treated with siRNA for Ang-1, Ang-2 or non-specific for 24 hours, and then we collected the supernatant after treatment with or without IFN/5-FU for 48 hours. We evaluated viability of HUVEC cells by MTT assay. The percentage of viable cells was significantly reduced by the conditioned media after the treatment of IFN/5-FU. There is no significant difference between the conditioned media after treatment with or without IFN/5-FU after knockdown of Ang-1 or Ang-2.