利用核磁共振造影分析蛋白質水解酶抑制劑 HAI-1在腫瘤生長與轉移中扮演之角色

Abstract

Hepatocyte growth factor/ scatter factor (HGF/ SF) is a pleiotropic cytokine which plays important roles in tumor growth and metastasis. To generate biologically active HGF, the conversion of single-chain HGF precursor (pro-HGF) to a heterodimeric form is essential. To date, several proteases including HGF activator (HGFA), factor XIIa, membrane-type serine protease-1 (MT-SP1)/matriptase, urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA) and hepsin have been supposed to involve in the activation of HGF. Among them, the HGFA exhibits the most potent activity. HGFA inhibitor type 1 (HAI-1), a type I transmenbrane protein, is a novel Kunitz-type serine protease inhibitor that inhibits HGFA, matriptase, hepsin and trypsin through its first Kunitz domain (Kunitz domain 1, KD-1). Thus, HAI-1 may play an important role in cancer progression and possess strong therapeutic potential. In this study, a recombinant secreted HAI-1 (sHAI-1) with KD-1 domain, fused to human IgG Fc region (sHAI-1-Fc) was constructed. The Fc portion of this fusion protein may enhance protein stability in vivo. Eukaryotic expression system was employed to produce the recombinant protein. By using conditioned medium containing sHAI-1-Fc, functional assays were performed. We demonstrated that sHAI-1-Fc fusion protein could inhibit the activity of trypsin and also have ability to bind matriptase. Further, the naked plasmid was introduced to mice by hydrodynamics-based gene delivery method. The recombinant proteins could be detected in serum of mice after administration. Thus, we may analyze the roles of sHAI-1 in tumor growth and metastasis in vivo in the future. In addition, experimental models for intrahepatic tumor inoculation and liver metastasis were set up by inoculating B16F10 mouse melanoma cell line into C57BL/6 mice. Furthermore, by prelabeling the cancer cells with iron oxide magnetic resonance contrast agent, we can monitor the tumor growth and cellular distribution by MRI detection. This preliminary study may provide a non-invasive tool for studies of cancer therapy.