Gene transfer of kringle 1-5 suppresses tumor development and improves prognosis of mice with hepatocellular carcinoma.
OBJECTIVE
Recent studies indicate that kringle 1-5 has a potent and specific antiangiogenic activity. Here, we investigated the antitumor effect of kringle 1-5 gene transfer on hepatocellular carcinoma in mice.


METHODS
The inhibitory effect of kringle 1-5 protein on proliferation of bovine capillary endothelial cells was evaluated by a tetrazolium-based assay. To study tumor growth, intrahepatic metastasis, and survival, liposome/kringle 1-5 complementary DNA complexes were injected intravenously in nude mice preimplanted with 1 of 3 hepatoma cell lines into the liver. Production of kringle 1-5 was tested by immunohistochemistry and Western blotting. Intratumoral vessel density was quantified. Expression of vascular endothelial growth factor, angiopoietin-1, and angiopoietin-2 in tumors was examined by Western blotting. Serum alanine aminotransferase and alpha-fetoprotein levels and body weights were measured.


RESULTS
Proliferation of bovine capillary endothelial cells was inhibited by purified kringle 1-5 in a dose-dependent manner. Gene transfer of kringle 1-5 caused a significant reduction in vessel density with suppression of tumor growth of the 3 hepatoma cell lines and serum alpha-fetoprotein levels, prolonged the survival period, and reduced the number of intrahepatic metastases. Among the analyzed angiogenic factors, kringle 1-5 reduced angiopoietin-2 expression levels. Expression of kringle 1-5 protein was detected on hepatoma cells and hepatocytes in the liver. However, it did not alter serum alanine aminotransferase levels and body weights, suggesting kringle 1-5 lacks severe side effects.


CONCLUSIONS
Antiangiogenic gene therapy with kringle 1-5 complementary DNA is a promising safe and effective strategy for suppression of growth of hepatocellular carcinoma.
Torimura
Takuji
T
Liver Cancer Division, Research Center for Innovative Cancer Therapy, and Center of the 21st Century COE Program for Medical Science, Second Department of Medicine, Fukuoka, Japan.
Ueno
Takato
T
Kin
Motoaki
M
Taniguchi
Eitaro
E
Nakamura
Toru
T
Inoue
Kinya
K
Sakata
Ryuichiro
R
Hashimoto
Osamu
O
Sakamoto
Masaharu
M
Ohira
Hiromasa
H
Kumashiro
Ryukichi
R
Sata
Michio
M
Yano
Hirohisa
H
Kojiro
Masamichi
M
Veitonmaki
Niina
N
Cao
Yihai
Y
D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D001835:Body Weight / Q000187:drug effects; D019556:COS Cells; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000235:genetics* / Q000473:pathology* / Q000503:physiopathology; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002522:Chlorocebus aethiops; D018076:DNA, Complementary; D042783:Endothelial Cells / Q000187:drug effects / Q000473:pathology; D018014:Gene Transfer Techniques; D006801:Humans; D018082:Kringles / Q000235:genetics*; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms / Q000098:blood supply / Q000235:genetics* / Q000473:pathology* / Q000503:physiopathology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009362:Neoplasm Metastasis / Q000517:prevention & control; D009389:Neovascularization, Pathologic / Q000473:pathology; D011379:Prognosis; D016019:Survival Analysis; D014162:Transfection
D018076:DNA, Complementary; D000410:Alanine Transaminase