| Angiogenesis,or neovascularization, the formation of new capillaries from preexisting blood vessels, is a critical step in the establishment,growth,and metastasis of solid tumors.Since Judah Folkman first proposed that all tumor growth and the subsequent development of metastases were dependment on the establishment of a new vascular supply in 1971 ,intensive efforts in many laboratories have been focused on finding potent anti-angio genesis agents with anti-tumor activity and on developing anti-tumor agents with a novel mechanish of aetion:to shut off delivery of nutrients,oxygen and growth factors.TNP-470,a synthetic analogue of the antibiotic fumagillin secreted by Aspergillus fumigatus,was discovered to be potent inhibitors of angiogenesis due to their inhibition of endothelial cell proliferation.In this study,cell culture and tumor- bearing nude mice were adopted to evaluate the efficacy of endothelial cell proliferation and tumor growth and microvascular angiogenesis by methods of flow cytometry,MTT,immunohistochomistry and stereology.The results were as follows: 1. In an MTT assay,after 72 hrs continuous exposure to TNP-470,growth inhibition was observed in pulmonary microvascular endothelial cell(PMVEC),LQVO cell line and PLCS7 cell line.The result showed that PMVEC was the most sensitive to TNP-470.The 50% inhibitory concentrations (IC50) was only 1 X 102ng/ml,LOVO cell line and PLC57 cell line were 5 X 1 O2ng/ml and 1 >( 1 O3ng/ml,respectively.These data showed LQVO cell line and PLC57 cell line were sensitive to TNP-470 only at doses that are 5 X i04 and 1 X lO~ times higher than the dose that inhibits PMVEC cell proliferation. 2. To determine if the inhibitory effects on PMVEC cell growth by TNP- 470 involved an arrest of the cell growth at a particular phase in the cell cycle,PMVEC cells were cultured with TNP-470 at lOng/mi for 48 hrs,and then the DNA content of the cells was measuerd by flow cytometric analysis.The results showed 7 1.9% proportion of the cells was found in the G0/Gl phase in -4- the treat group,but the control group was 58.7%.The results indicated that TNP- 470 could affect endothelial cells cycle. So TNP-470 caused an increased proportion of cells in G0/Gl phases and a decreased proportion in G2/M and 5- phases. 3. The effect of TNP-470 on tumor growth was evaluated by tumor-bearing nude mice modeal.BALB/c nude mice were injected subcutaneously with LQVO cells and PLC57 cells and randomized into a treatment group and a control group.In the treatment group,TNP-470 was injected 30 mg/kg subcutaneously every other day,while the vehicle(three per cent enthanol solution in 0.9% per cent saline)was give to the mice in control group.Results:At day 30 of treatment ,in LOVO cell line,tumor volume(mm3) was 478.57 ?309.67 in the control group versus 1360.42?18.26 in the TNP-470 treated group,and T:C ratio (mean tumor volume of treat mean tumor volume of control) was 1:2.8, resulting in a 65% percent decrease in tumor growth.But in PLC57 cell line,central necrosis and consequential shrinkage of tumors occurred in treatment and control groups.At the end of experiment,there was no significant difference in tumor volume between the two groups. 4. The effect of TNP-470 on suppression of microvascular angiogenesis in the subcutaneously implanted LOVO cell line was assessed by immunohistochemical staining and stereological an... |
PDF Full Text Request