1. Volume 130, Issue 4, Pages 1301-1310 (April 2006)
Gene Transfer of Kringle 1–5 Suppresses Tumor Development and Improves Prognosis of Mice With Hepatocellular Carcinoma 
Takuji Torimura, Takato Ueno, Motoaki Kin, Eitaro Taniguchi, Toru Nakamura, Kinya Inoue, Ryuichiro Sakata, Osamu Hashimoto, Masaharu Sakamoto, Hiromasa Ohira, Ryukichi Kumashiro, Michio Sata, Hirohisa Yano, Masamichi Kojiro, Niina Veitonmaki, Yihai Cao 
Gastroenterology 
Volume 130, Issue 4, Pages (April 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

2. Figure 1
(A) Secretion of K1–5 protein into the media of pcDNA3.1/K1–5 cDNA–transfected Cos-1 cells. Thirty-six hours after transfection, Cos-1 cells were incubated in serum-free media for 24 hours. Secretion of K1–5 protein into the media was confirmed by Western blotting with anti-His prove antibody. (1) Media of Cos-1 cells transfected with pcDNA3.1/K1–5 cDNA. (2) Media of Cos-1 cells transfected with pcDNA3.1. (B) Expression of endogenous K1–5 protein by human hepatoma cells. (1) KYN-2. (2) KIM-1. (3) HAK-1 B. (C) Inhibitory effects of purified K1–5 on BCE cells. BCE cells were cultured with 100 μL of the media containing 0, 1, 10, and 100 μmol/L K1–5, 5% FBS, and 0.1 ng fibroblast growth factor 2. Seventy-two hours after incubation, cell proliferation was evaluated by a tetrazolium-based assay. Data are shown as a ratio of the control and are expressed as mean ± SD of 12 samples. *P < .001 by Kruskal–Wallis test.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

3. Figure 2
Inhibition of tumor growth in the liver of nude mice by injection of liposome/pcDNA3.1/K1–5 cDNA complexes. Liposome-DNA complexes or PBS was injected via the tail vein. Twenty-eight days after tumor cell implantation, mice were killed and tumor volume and serum AFP levels were measured. (A) Representative photographs of the liver. (1) An enlarged tumor of KYN-2 cells is evident in the circle of liver injected with liposome-pcDNA3.1 complexes. (2) No tumor is present in the liver injected with liposome/pcDNA3.1/K1–5 cDNA complexes. (B) Tumor volumes are expressed as mean ± SD (n = 6 per group). (1) Tumor volume of mice implanted with KYN-2 cells. **P = .001 by Kruskal–Wallis test; n.s., not significant by Mann–Whitney U test compared with pcDNA3.1. (2) Tumor volume of mice implanted with KIM-1 cells. *P = by Mann–Whitney U test compared with pcDNA3.1. (3) Tumor volume of mice implanted with HAK1-B cells. *P = by Mann–Whitney U test compared with pcDNA3.1. (C) Serum AFP levels are expressed as mean ± SD (n = 6 per group). *P = by Kruskal–Wallis test.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

4. Figure 3
Prolonged survival period of KYN-2–implanted mice by injection with liposome/pcDNA3.1/K1–5 cDNA. The survival period was counted from the day of tumor cell transplantation. Data are expressed as mean ± SD (n = 6 per group). *P = .008 by Mann–Whitney U test compared with pcDNA3.1.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

5. Figure 4
Inhibition of intrahepatic metastasis in severe combined immunodeficient mice implanted with KYN-2 cells by injection of liposome/pcDNA3.1/K1–5 cDNA complexes. The mice were killed and the number of tumor nodules was counted after 28 days of KYN-2 cell implantation. (A) Representative photographs of the liver. (1) Numerous tumor nodules of KYN-2 cells were observed in the liver of mouse injected with liposome-pcDNA3.1 complexes. (2) Only 1 tumor nodule was detected in the liver of mouse injected with liposome/pcDNA3.1/K1–5 cDNA complexes. (B) Numbers of tumor nodules in the liver are expressed as mean ± SD (n = 6 per group). *P = .006 by Mann–Whitney U test compared with pcDNA3.1.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

6. Figure 5
Expression of K1–5 protein in the liver of mice injected with liposome/pcDNA3.1/K1–5 cDNA complexes. (A) Immunohistochemical analysis of K1–5. (1) In a tumor nodule of KYN-2 cells, immunoreactive products of anti-His prove antibody were detected in the cytoplasm of hepatoma cells (arrows). (2) In noncancerous liver tissue of mice transplanted with KYN-2 cells, immunoreactive products of anti-His prove antibody were detected in the cytoplasm of hepatocytes (arrows) (original magnification 200×). (B) Western blot analysis of K1–5. Production of K1–5 protein in tumor tissues and noncancerous liver tissue was confirmed by Western blotting with anti-His prove antibody. (1) Tumor tissue. (2) Noncancerous liver tissue.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

7. Figure 6
Reduced vascularization in tumor tissue of mice injected with liposome/pcDNA3.1/K1–5 cDNA complexes. The density of CD31-positive vessels in a tumor field is represented as mean ± SD (n = 50 per group). *P < by Mann–Whitney U test compared with pcDNA3.1-treated group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

8. Figure 7
Changes in VEGF, Ang-1, and Ang-2 expression in tumor tissue by injection of liposome/pcDNA3.1/K1–5 cDNA complexes. (A) Western blot analysis of VEGF, Ang-1, and Ang-2 in tumor tissue of KYN-2 cells. Lanes 1–6, tumor tissues treated with pcDNA3.1/K1–5 cDNA; lanes 7–12, tumor tissues treated with pcDNA3.1 (1), VEGF (2), Ang-1 (3), Ang-2 (4), Actin. (B) VEGF 165, Ang-1, and Ang-2 proteins were quantitated by densitometry and normalized against the density of actin. Data indicate the ratio compared with the pcDNA3.1-treated group. Data are expressed as mean ± SD of 6 samples, respectively. *P = .007 by Mann–Whitney U test compared with pcDNA3.1.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

9. Figure 8
Influence of injection of liposome/pcDNA3.1/K1–5 cDNA complexes on serum ALT levels. Serum ALT levels of mice were measured by enzyme-linked immunosorbent assay. Data are mean ± SD of 6 samples. *P = by Kruskal–Wallis test.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions