Gene Transfer of Kringle 1–5 Suppresses Tumor Development and Improves Prognosis of Mice With Hepatocellular Carcinoma

Background & Aims: 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 α-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 α-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.

Abbreviations used in this paper:  AFP, α-fetoprotein , Ang, angiopoietin , BCE, bovine capillary endothelial , CAT, chloramphenicol acetyltransferase , DOTAP, dioleoyl trimethylammonium propane , FBS, fetal bovine serum , His, hexahistidine , HRP, horseradish peroxidase , K, kringle , VEGF, vascular endothelial growth factor