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


CONCLUSIONS
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.
Wada
Hiroshi
H
Department of Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka E-2, Suita 565-0871 Osaka, Japan. hwada98@gesurg.med.osaka-u.ac.jp
Nagano
Hiroaki
H
Yamamoto
Hirofumi
H
Noda
Takehiro
T
Murakami
Masahiro
M
Kobayashi
Shogo
S
Marubashi
Shigeru
S
Eguchi
Hidetoshi
H
Takeda
Yutaka
Y
Tanemura
Masahiro
M
Umeshita
Koji
K
Doki
Yuichiro
Y
Mori
Masaki
M
D042501:Angiogenic Proteins / Q000235:genetics / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D015536:Down-Regulation; D004357:Drug Synergism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005472:Fluorouracil / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology
D042501:Angiogenic Proteins; D016898:Interferon-alpha; D005472:Fluorouracil