| Tumor is one of the primary diseases to theaten human helth in developing and developed countries. The need for the development of new antitumor drugs is evident and of great importance for the future. Today tumor chemotherapy depends on a rather small panel of compounds from which a major part are in clinical use since more than 20 years. With the enormous body of knowledge which has been accumulated in chemistry, cell and molecular biology in the last few years it has become possible to develop novel molecules which, for example, exert their cytotoxic actions on cell membranes and on signal transduction pathways. The preparation of antitumor drugs is a important strategy against tumor diseases. Due to the complexity of the development of tumor, ploy-function drug therapies will become one of promising way to overcome the tumor diseases.Studies have shown that tumor growth is dependent on angiogenesis. Basement membranes are thin layers of a specialized extracellular matrix that provide the supporting structure on which epithelial cells grow and that surround mechanical support but also influence cellular behaviors such as differentiation and proliferation. The one of major macromolecular constituents of basement membranes is type IV collagen. In general, type IV collagen promotes cell adhesion, migration, differentiation, and growth. The a3(IV) NC1 domain has been shown to inhibit the proliferation of capillary endothelial cells and blood vessel formation using in vitro and in vivo models of angiogenesis and in inducing endothelial cell-specific apoptosis. Both of them provide the rationale for antiangiogenic and cytotoxic therapy in tumor. The NC1 domain of a3 chain of type IV collagen, namely tumstatin, has been shown to displayspecific anti-angiogenic properties by inhibiting endothelial cells' proliferation and inducing their apoptosis via an interaction with av(33 integrin. Through a requisite interaction with av(33 integrin, tumstatin inhibits activation of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3-kinase), protein kinase B (PKB/Akt), and mammalian target of rapamycin (mTOR), and it prevents the dissociation of eukaryotic initiation factor 4E protein (eIF4E).Citropin 1.1, a broad-spectrum antibiotic and antitumor agent, is a 16 residual peptide which is isolated from skin secretions of the skin glands of the tree frog, L. citropa. Studies of citropin 1.1 show that the peptide adopt conventional amphipathic a-helical structures at N-terminal, a feature commonly found in membrane-active agents. Interaction occurs at the membrane surface with the charged, and normally basic peptide adopting an a-helical conformation and attaching itself to charged, and normally anionic sites on the lipid bilayer. This selectively causes disruption of normal membrane function leading to lysis of the tumor cells which have significantly higher levels of anionic phospholipids present in the outer leaflet.Citrostatin, a fused antitumor peptide was designed, synthesized and purified and its human tumour line test (anticancer) and antiangiogenic functions were tested. The fused peptide was composed of two inhibitory regions: (1) T8 peptide, a deletion mutant from tumstatin (69-95 amino acids. Leu at position 69 mutated to Lys, the NCI domain of a3 chain of type IV collagen), possesed anti-angiogenic activity, inhibited proliferation and induced apoptosis specifically in endothelial cells. (2) citropinl.18, synthetic modification of citropin 1.1, which has a variety of anticancer activities. This 48-amino acid-fused peptide was artificially synthesized, purified and analysed. The purpose of this research was to establish techniques for preparation of fused citrostatin and to study the biological activities of it in vitro and in vivo. This study was divided into three steps as follow:1) Design, synthesis, purifying and characterization of citrostatin peptide.2) Construction of the expression vector containing the fusion gene encoding citropinl.18 and T8 (tumstatin 69-95).3) Prokaryotic expression of fusion gene citrostatin and activity mesurement of fusion peptide citrostatin in vitro and in vivo.This study finally showed that gene citropinl.18 and T8 were fused successfully into prokaryotic expression vector pMAL-c2X. The peptide citrostatin and MBP/citrostatin had the similar activities of antitumor as well as antiangiogenesis. Cytotoxicity was measured by checking ED50 values with the MTT assay. These peptides could inhibit the proliferation of endothelial cell ECV304 with significantly difference (P<0.05) and this difference become more marked (P<0.01) with increase of concentration. Data were examined by use of linear regression estimate that ED5o values of ECV304 cell treated with citrostatin and MBP/citrostatin are 6.2^g/ml and 2.6x10"6 mol/L. These peptides also could inhibit the proliferation of tumor cells (1990, NCI-H640 and A375) with significantly difference (P<0.05) and this difference become more marked (P<0.01) with increase of concentration. Treated with citrostatin, the corresponding ED50 values of 929 and NCI-H640 cells are 50ug/mL 16ug/ml. Treated by MBP/citrostatin, the corresponding ED50 values of 1990, NCI-H640, A375 cells are 2.2xlO"5mol/L, 1.2><10"5 mol/L, 1.6xl0"5 mol/L. These results suggest that our design on citrostatin gene was successful, and the bifunctional peptide will useful to human tumor therapy with its antiangiogenic and antitumor activities.
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