Direct and indirect effects of IFN-α/5-FU on HUVEC

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, 25 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 30 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 35 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 40 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 Direct and indirect effects of IFN-α/5-FU on HUVEC Wada H, et al., Page 3 regulation of angiogenic factors released from HCC cells. Modulation of VEGF and 45 Angs secretion by IFN-alpha and 5-FU may contribute to their anti-angiogenic and anti-tumor effects on HCC.