The kringle domain of tissue-type plasminogen activator inhibits in vivo tumor growth Byoung-Shik Shim a,1 , Byoung-Hak Kang a,1 , Yong-Kil Hong a,1 , Hyun-Kyung Kim a , Il-Ha Lee b , Soo-Young Lee b , Young-Joon Lee c , Suk-Keun Lee c , Young Ae Joe a, * a Cancer Research Institute, Catholic Research Institutes of Medical Sciences, The Catholic University of Korea, Seoul 137-701, Republic of Korea b Department of Natural Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea c Department of Oral Pathology, College of Dentistry, Kangnung National University, Kangnung, Kangwon-Do 210-702, Republic of Korea Received 17 December 2004 Available online 31 December 2004 Abstract The two-kringle domain of tissue-type plasminogen activator (t-PA) has previously been shown to contain anti-angiogenesis activity. In this study, we explored the potential in vivo anti-tumor effects of the recombinant kringle domain (TK1-2) of human t-PA. Anti-tumor effects of purified Pichia-driven TK1-2 were examined in nude mice models by subcutaneous implantation of human lung (A-549) and colon (DLD-1, HCT-116) cancer cell lines. Mice bearing the tumors were injected with PBS or purified TK1-2 (30 mg/kg) i.p. every day for 22 days. TK1-2 treatment suppressed the A-549, DLD-1, and HCT-116 tumor growth by 85.3%, 52.4%, and 62.5%, respectively. Immunohistological examination of the tumor tissues showed that TK1-2 treatment decreased the vessel density and also the expression of angiogenesis-related factors including angiogenin, VEGF, a-SMA, vWF, and TNF-a, and increased the apoptotic fraction of cells. TK1-2 neither inhibited in vitro growth of these cancer cells nor affected t-PA-mediated fibrin clot lysis. These results suggest that TK1-2 inhibits the tumor growth by suppression of angiogenesis without interfering with fibrinolysis. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Tumor; Angiogenesis; Tissue-type plasminogen activator; Kringle; Inhibitor Angiogenesis, the process of new capillary outgrowth from pre-existing vessels, is essential for embryonic development, organ formation, wound healing, female reproduction, tissue regeneration, and remodeling [1,2]. Under pathological conditions such as tumorigenesis and diabetic retinopathy, switching to an angiogenic phenotype occurs by up-regulation of angiogenic factors and perhaps down-regulation of angiogenesis inhibitors [3–5]. Cancer cells begin to promote angiogenesis early in tumorigenesis. Many recent studies indicate tumor growth and metastasis depend on angiogenesis [6–11]. Several studies also suggest angiogenesis is regulated by a balance of proteins that promote and suppress angiogenesis, which has encouraged the use of angiogen- esis inhibitors as a promising therapeutic strategy for preventing angiogenesis in cancer. Among endogenous angiogenesis inhibitors, angio- statin—which was isolated from serum and urine of tu- mor-bearing animals—consists of the first four kringle domains of plasminogen [6]. In vivo, angiostatin sup- presses neovascularization and tumor growth in animals without displaying toxic effects [6,12]. Kringle domains are protein modules composed of 78–80 amino acids connected by a characteristic triple disulfide-linked loop. The triple disulfide bonds are strictly conserved between kringles [13]. These domains appear to be independent 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.12.126 * Corresponding author. Fax: +82 2 532 0575. E-mail address: youngjoe@catholic.ac.kr (Y.A. Joe). 1 Equal contribution was made by these authors. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 327 (2005) 1155–1162 BBRC