Combination therapy of interferon-alpha and 5-fluorouracil inhibits tumor angiogenesis in human hepatocellular carcinoma cells by regulating vascular endothelial growth factor and angiopoietins.
We recently reported that interferon-alpha (IFN-alpha) and 5-fluorouracil (5-FU) combination therapy in advanced hepatocellular carcinoma (HCC) achieved excellent clinical results. However, the mechanism underlying this combination therapy remains to be elucidated. In this study, we examined the anti-tumor effects of IFN-alpha and 5-FU combination therapy in vivo and aimed to reveal its anti-angiogenic effects by investigating the expression of vascular endothelial growth factor (VEGF) and angiopoietins (Ang-1 and Ang-2). Human HCC cells, HuH7, were subcutaneously injected in nude mice. Ten days later, groups of mice received treatment with IFN-alpha alone, 5-FU alone, or with a combination of IFN-alpha and 5-FU for four weeks. Immunohistochemical examinations of proliferating cell nuclear antigen (PCNA), cell differentiation antigen 34 (CD34), Ang-1, -2 and VEGF, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and quantification of VEGF, Ang-1 and-2 mRNA using real-time RT-PCR were performed. Results showed that IFN-alpha and 5-FU combination therapy significantly inhibited the growth of human HCC cells compared with the control group or single agent treatment. The combination therapy decreased PCNA-positive cells as well as microvessel density (MVD) and induced apoptosis of (TUNEL-positive cells) more than other treatment groups. Immunohistochemical analysis revealed that the combination therapy significantly decreased the expression of VEGF and Ang-2 and increased that of Ang-1. The ANG2/ANG1 mRNA expression ratio was significantly lower in the combination therapy group. In conclusion, our results suggested that IFN-alpha and 5-FU combination therapy has anti-proliferative and anti-angiogenic effects and can induce apoptosis in vivo. The synergistic and anti-angiogenic effects may in part be attributable to the regulation of Ang-1, -2 and VEGF.
Wada
Hiroshi
H
Department of Surgery, Graduate School of Medicine, Osaka University, Suita 565-0871, Osaka, Japan.
Nagano
Hiroaki
H
Yamamoto
Hirofumi
H
Arai
Isao
I
Ota
Hideo
H
Nakamura
Masato
M
Damdinsuren
Bazarragchaa
B
Noda
Takehiro
T
Marubashi
Shigeru
S
Miyamoto
Atsushi
A
Takeda
Yutaka
Y
Umeshita
Koji
K
Doki
Yuichiro
Y
Dono
Keizo
K
Nakamori
Shoji
S
Sakon
Masato
M
Monden
Morito
M
D042682:Angiopoietins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000098:blood supply*; D004357:Drug Synergism; D005260:Female; D005472:Fluorouracil / Q000008:administration & dosage; D006801:Humans; D007124:Immunoenzyme Techniques; D016898:Interferon-alpha / Q000008:administration & dosage; D008113:Liver Neoplasms / Q000098:blood supply*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009389:Neovascularization, Pathologic / Q000188:drug therapy*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*