Inhibitory Effect And Mechanism Of Endostatin On The Angiogenesis Of Xenografted Namalwa Lymphoma In Nude Mice

Objects:1.To investigate the inhibitory effect of endostatin on the growth and angiogenesis ofxenografted Namalwa lymphoma in nude mice.2.To investigate the relationship between regulated Foxol protein expression anddecreased angiogenesis in xenografted Namalwa lymphoma by endostatin.Methods:1.Xenografted Namalwa lymphoma in nude mice was generated by subcutaneouslyimplanted Namalwa cells.And they were divided to five groups.Injections were givenintraperitoneally.Recombinant human endostatin was given at doses of2.5mg/kg/d,5mg/kg/d and 10mg/kg/d respectively.Negative control group was givensaline with same volume,and positive control group was given with cyclophosphamide(CTX) at the dose of 75mg/kg every two days for 3 times.Tumor volume wasmeasured with calipers,and the volume was calculated with the formula:width~2×lenth×0.52,as described previously.Relative tumor volume (RTV) and T/C%were also calculated.The endpoint was set when the RTV of any endostatin-treatedgroup was smaller than the saline-treated group significantly.2.Mouse microvessel endothelial celles were cocultured with 0μg/ml,20μg/ml and80μg/ml endostatin for 16 hours.The effect of endostatin on the migration ofendothelial cell was studied by transwell,and the proliferation was observed by XTT.Foxo 1 expression was studied by RT-PCR and western blot.3.Xenografted Namalwa lymphomas in nude mice were generated and given with salineor endostatin in protocol on the base of in vivo study.Tumor tissue was separated afterthe treatment,and was digested with collagenase I to single-cell suspension.Microbeads were used to seperate CD31 positive cells from the suspension.The Foxolprotein expression of CD31 positive cells was compared between endostatin- andsaline-treated groups.Results1.Xenografted Namalwa lymphoma occurred in 100% nude mice.No time difference wasfound when the tumor was palpable (P=0.523).And no mice died during theexperiment.2.No RTV difference was found among 5 groups after 6-days treatment (P=0.185).RTVof CTX-treated group was smaller than that of saline-treated group after 8-daystreatment (P=0.003).RTV of 5mg/kg/d endostatin-treated group was smaller than that of saline-treated group after 12-days treatment (P=0.027),and T/C% was 64.2%.Nodifference in RTV was found between the other two endostatin- and saline-treatedgroups,and T/C% was 87.4% and 85.9% respectively.3.The RTV of 5mg/kg/d group was larger than that of CTX group after 12-days treatment(P=0.046),and the T/C% of the latter is 38.4%.4.MVD in 5mg/kg/d group was lower than that in saline group after treatment for 12consecutive days,and the difference was significant(P=0.006),while MVD in2.5mg/kg/d and 10mg/kg/d groups were not significantly different from that in salinegroup。5.The number of endothelial cells passing the memberance decreased when treated withendostatin for 16 hours(P=0.000),and no effect was found on OD value of mousemicrovessel endothelial cell(P=0.484)6.The mRNA and non- phosphorylated Foxo 1 protein was upregulated by endostatin atthe same time(P＜0.05).7.CD31 positive cells of suspension from xenografted Namalwa lypmphoma accountedfor 17.2% before separation and 90.9% after separation.8.Endothelial Cells separated from endostatin-treated xenograted Namalwa lymphoma bymagnetic bead had more non-phosphorylated Foxol expression than those fromsaline-treated ones(P=0.047).But the CD31 negative cell had no Foxol expression(P=0.896)Conclusions:1.The growth of xenografted Namalwa lymphoma could be inhibited even if the tumorburden was prominent.2.The exact dose was necessary to inhibit the growth of xenograted Namalwa lymphoma.Higher or lower dose than the definite dose range was less effective.3.The inhibition on the growth of xenograted Namalwa lymphoma was related todecreased angiogenesis.4.Endostatin could inhibit the migration of mouse microvessel endothelial cell.Sincemigration was one of the most important steps in angiogenesis,it was one of theimportant ways in which endostatin decreased angiogenesis.5.There was common trend between inhibited migration and upregulated non-phosphorylated Foxol expression of endothelial cells treated by endostatin.And sothere may be relationship between them.6.Endostain raised non-phosphorylated Foxol expression,and at the same time endostatin decreased angiogenesis.Up-regulation of non-phosphorylated Foxolexpression was one of endostatin's mechanisms.