| Background and objectivesEsophageal carcinoma is one of the most lethal gastrointestinal malignancies that generate on epithelial tissue of esophagus. Its major pathology is squamous cell carcinoma. The cause of esophageal carcinoma has association with diet habit, genetic factors and so on, but the definite cause is still unclear. There is high incidence of esophageal carcinoma in China. Due to fast growth, easy transfer and poor prognosis, the overall 5 year survival of esophageal cancer is still very low in spite of treatment with surgery, chemotherapy, radiotherapy or a combination of these treatment modalities. so there is a pressing need for new direction in the management of esophageal carcinoma.In 1971, Dr.Folkman first proposed the hypothesis that tumor growth is angiogenesis dependent, This theory has been proved to be right later.The diffusion capacity for oxygen and nutrients in tissues is limited to about 150-200μm, therefore most cells of an organism are located with in this radius around a capillary. The rapid growth of tumors required the formation of new blood vessels to provide adequate nutrition, tumor achieve its growth rely on excessive angiogenesis. Tumor will keep dormant or regress without neovascularization, Therefore interfering with this process poses for controlling tumor growth means a new research direction. Thus tumor growth depends on balance between circulating endogenous proangiogenic factors and endogenous angiogenesis inhibitors (Arresten,Canstatin, Tumstatin). through the human intervention on expression of angiogenesis inhibitor and pro-angiogenic factors, we can effectively inhibit tumor angiogenesis, tumor growth and metastasis.Arresten, Canstatin, Tumstatin and Endostatin were isolated from Extracellular matrix,they are the NC1 domain of the collagen which were previously considered non-functional area, They were proved to have the capacity of inhibiting tumor growth. Arresten has been proved can efficient inhibit angiogenesis and tumor growth in mouse models, At the same time, Arresten significantly increases apoptosis of endothelial cells in vitro by decreasing the amount of anti-apoptotic molecules of the Bcl-family(Bcl-2 and Bcl-xL).The protein Endostatin, a 20kD proteolytic fragment of typeⅩⅧcollagen, is one of the most active natural inhibitors of angiogenesis. Endostatin specifically inhibits the in vitro and in vivo proliferation of endothelial cells, inducing their apoptosis through inhibition of cyclinD1. Endostatin is a potent inhibitor of angiogenesis. Fibroblast growth factor-2(bFGF) induced angiogenesis in the chicken chorioallantoic membrane was inhibited by endostatin, tumstatin is the noncollagenous (NC1) domain of the alpha 3 chain of typeⅣcollagen, it inhibits in vitro melanoma cell proliferation and migration. It was demonstrated that the overexpression of Tumstatin induced an in vivo down-regulation of proteolytic cascades involving matrix metalloproteinases (MMPs), especially the production or activation of MMP-2, MMP-9, MMP-13.Canstatin is the 24 kDa NC1 domain of the a2 chain of typeⅣcollagen,and human Canstatin was recombinantly produced in E.coli and 293 human embryonic kidneycells. Canstatin inhibits the proliferation of human endothelial cells in a dose-dependent manner and induces apoptosis, and it has no effect on non-endothelial cells. Canstatin can inhibit the growth of a variety of tumors, at present,the mechanism of this is unclear, it was considered that had closely relation with inducing cell proliferation, migration and induction of apoptosis.The inhibiting effect of Canstatin on angiogenesis is comparatively more stronger than the similar factors available at present, hence, canstatin has a higher research value and a wider prospect in clinical application, for which there is necessary to make further study. This study will approach the inhibiting effect of human canstatin gene on human esophageal carcinoma xenografts.Materials and MethodsThere are 21 female BALB/c nude mice,4-5 weeks old, weighing 16-20g, KYSE150 cells was transplanted subcutaneously to establishe xenograft human esophageal carcinoma model in nude mice. When the tumor grew to 50mm3, nude mice with subcutaneous were randomly divided into three groups when size of tumor was 50mm3. Ad-GFP-canstatin was experimental group, adenovirus green fluorescent protein (Ad-GFP)and PBS groups were control groups.The size of subcutaneous tumor was measured and recorded by every 3 days during treatment. After 30 days, the mice were killed to observe tumor size. The expression of bcl-x1,cyclin D1,bFGF and MMP-13 were measured by RT-PCR (Reverse Transcriptase).ResultsAfter first week of transfection,Compared with that in Ad-GFP and PBS groups, tumor volume was significantly suppressed in Ad-GFP-canstatin group. The inhibition rate of tumor growth was up to 65% at the ninth day. Compared with those in Ad-GFP and PBS groups, the expressions of bcl-x1,cyclin D1,MMP-13 in canstatin group was higher (p<0.05), The difference has obvious statistically significant.but the difference between Ad-GFP and PBS groups has no statistically significan(p>0.05). but the difference of the expressions of bFGF has no statistically significant among three groups.ConclusionsNude mice is an ideal animal model for tumors transplantation. Subcutaneous esophageal carcinoma in nude mice is easily achieved. And it was easily operated and achieved to establish subcutaneous esophageal carcinoma in nude mice with cell suspension method. It also shows that canstatin can inhibit the growth of human esophageal carcinoma and the mechanism is to inhibit the expression of bcl-xl, cyclin D1, MMP13, these can provid assistance to further study on anti-angiogenesis effect and clinical application of human canstatin gene. |
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