| Background The process of cancer metastasis consists of a series of sequential steps, all of which must be successfully completed to give rise to new metastatic foci.Folkman noted in the 1970s that blood supply was necessary for tumor growth,metastasis and . Tumors will stop growing or die when it exceeds 2 to 3 mm in diameter if new blood vessels are not formed. Numerous studies have also shown that inhibition of tumor growth could be achieved by inhibiting angiogenesis. Moreover, genes for endothelial cells, targeted by antiangiogenic factors, have stable genetic properties and a low mutation rate. As a result, acquired drug resistance to antiangiogenic factors rarely developed during antiangiogenic therapy. These results point to anti-angiogenesis as being one of the most effective and promising ways to cure and control the development of tumors in the future.The development of new blood vessels from preexisting ones is generally referred to as angiogenesis. In the adult, new blood vessels arise via angiogenesis,a process critical foe normal physiological events such as wound repair, the menstrual cycle, and endometrium remodeling. In the last decades, considerable research has been conducted documenting that tumor growth and metastasis repair angiogenesis. This process is pivotal to the survival and subsequent growth of solid tumors beyond a few cubic millimeters in size. Vascular basement membrane constitutes an insoluble structural wall of newly formed capillaries and undergoes several changes during tumor-induced angiogenesis. Initially, the membrane is degraded and disassembled but is finally reorganized to a native state around a newly formed capillary. Such vascular matrix changes during angiogenesis are associated with the expression of matrix proteins that can interact with vascular endothelium and provide endogenous angiogenic and anti- angiogenic signals. Basement membranes are composed of macromolecules such as typeⅣcollagen, laminin, HSPG, fibronectin, and entactin. typeⅣcollagen is composed of six genetically distinct gene products, namely,α1-α6.theα1 andα2 isoforms are ubiquitously present in human basement membranes. The other four isoforms exhibit restricted distributions. TypeⅣcollagen promotes cell adhesion, migration, differentiation, and growth. It is thought to play a crucial role in endothelial cell proliferation and behavior during the angiogenic process. Several studies have shown the anti- angiogenic properties with inhibitoes of collagen metabolism, supporting the notion that basement membrane collagen synthesis and deposition are crucial for blood vessel formation and survival. Additionally, the COOH-terminal globular NC1 domain of typeⅣcollagen is speculated to play an important role in the assembly of typeⅣcollagen suprastructure, basement membrane organization, and modulation of cell behavior. Recently, the NC1 domain ofα2 chain of typeⅣcollagen (canstatin) was identified as an angiogenesis inhibitor. In the present study, we demonstrate the pivotal role of arresten, the NC1 domain ofα1 chain of typeⅣcollagen, in modulating the function of capillary endothelial cells and blood vessel formation using in vitro and in vivo models of angiogenesis and tumor growth..In this report, we identify an important anti-angiogenic vascular basement membrane-associated protein, the 26-kDa NC1 domain of theα1 chain of typeⅣcollagen, termed arresten. Arresten was isolated from human placenta and produced as a recombinant molecule in Escherichia coli and CHO cells. We demonstrate that arresten functions as an anti- angiogenic molecule by inhibiting endothelial cell proliferation, migration, tube formation, and Matrigel neo-vascularization. Arresten inhibits the development of tumor metastases in nude mice. Additionally, we show that the anti-angiogenic activity of arresten is protentially mediated via mechanisms involve cell surface proteoglycans and theα1β1 integrin on endothelial cells. Collectively, our results suggest that arresten is a potent inhibitor of angiogenesis with a potential for therapeutic use.Object To evaluate the specifically inhibition tumor metastases of arresten anti- angiogenic activity in huvec and nude mice and its mechanisms.Methods By gene transfected way, pSecTag2-arresten was transfected into CHO cell line ,the expression of arresten was measure by PCR,Western-blot, now arresten protein was determinated .Then the result of arresten inhibited of huvec proliferation, migration, tube formation, and Matrigel neo-vascularization was searched by MTT,cell migration and anchor to three-dimensional vascular structures. The correlation mechanism of arresten molecule biology was discussed by PCR,Western-blot. Moreover the inhibition of arresten of LOVO cell adhesive huvec was probed. Then in the nude mice pSecTag2-arresten was transfected into human colonic cancer cell line LOVO using Lipofectamine 2000.RT-PCR and Western-blot method was used to examine the expression of arresten mRNA and arresten protein, the biological behaviors of genetically modified LOVO cell clone was further investigated with MTT. At last, the LOVO cell expressing arresten were implanted into nude mice to investigate the prevention of hepatic metastasis of colon cancer, and the weights of tumor were recorded and analyzed,in the cancer ,MVD was detected by immunohistochemistry.Result Arresten gene was expressed successfully in transfected CHO cell, and the positive CHO cell clones expressing human arresten gene stable obtained, it can offer arresten protein. Arresten can inhibit huvec proliferation, migration, tube formation, and Matrigel neo-vascularization by inhibiting the expression of VEGF. Moreover it can inhibit the adhesive of LOVO cell to huvec by inhibiting expression of VCAM-1. The LOVO cell line with the character of expression of arresten was obtained, the biological behaviors of it was not modified. Both the MVD and the growth of colonic cancer with hepatic metastasis in nude mice of LOVO cell transfected arresten gene were suppressed.Conclusion Arresten can inhibit angiogenesis and adhesive of tumor cell to huvec, by these ways it can inhibit tumor metastasis. It is hoped to be a new therapeutic agent for treating the metastasis of tumor.
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