Inhibitory Effect and Mechanism of Endostatin on the Angiogenesis of Xenografted Namalwa Lymphoma in Nude Mice
|Keywords||Endostatin Burkitt's lymphoma Angiogenesis inhibitors Forkhead box protein O1|
Objective 1. Learn endostatin Namalwa lymphoma in nude mice transplanted tumor growth and tumor angiogenesis inhibition. 2. Preliminary study endostatin reduce Namalwa lymphoma the transplanted tumor angiogenesis and regulation of the expression of vascular endothelial cell forkhead box protein O1 relationship. Method 1. In vivo: subcutaneous injection of Namalwa lymphoma cells to establish nude mouse model. 11 days after tumor explants grouping intervention, daily intraperitoneal injection endostatin 2.5mg/kg, 5mg/kg and 10mg/kg; positive control group was injected with cyclophosphamide 75mg/kg, every two days times, a total of 3 times; negative control group daily injections of the same volume of normal saline. Once every two days measured maximum vertical diameter of tumor, tumor volume was calculated length × width ~ 2 × 0.52, each group was compared relative tumor volume (tumor volume after the intervention / intervention before the tumor volume), and calculate the relative tumor proliferation rate. Any the endostatin group antitumor effect appears to stop interfering killed nude mice bearing isolated tumor endothelial cells CD31 immunohistochemical staining and microvessel density (MVD). 2. Vitro: mouse microvascular endothelial cells were and 0μg/ml, 20μg/ml and 80μg/ml endostatin in vitro co-culture 16 hours observation cell migration using transwell chambers XTT assay cell proliferation activity, RT-PCR and Determination of cell forkhead box protein O1 western blot expression at the mRNA and protein levels. 3. Vitro experiments: the establishment of Namalwa lymphoma xenografts in nude mice were injected with an effective dose of endostatin (determined according to the first part of the study intervention programs) and saline intervention to end the separation of tumor tissue using magnetic bead sorting CD31 marker vascular endothelial cells, and compare their forkhead box protein 01 expression levels. Results: 1. The transplanted tumor formation rate of 100%, each composed of tumor time difference was not statistically significant (P = 0.523) during the intervention groups of nude mice did not occur to death. 2. 6 days after the intervention, each group relative tumor volume difference was not statistically significant (P = 0.185); eight days after intervention the cyclophosphamide group relative tumor volume less than the saline group (P = 0.003); 12 days after the intervention, 5mg/kg/d endostatin group relative tumor volume less than the saline group (P = 0.027), the tumor relative growth rate of 64.2%; and 2.5mg/kg/d, 10mg/kg/d group and no statistically significant differences in the saline group, P = 0.693 and 0.604, respectively, the tumor relative growth rates were 87.4% and 85.9%. 3. Intervention 12 days 5mg/kg/d endostatin group relative tumor volume greater than the cyclophosphamide group (P = 0.046), the latter tumor relative growth rate of 38.4%. 4. 12 days after the intervention, the tumor tissue of 5mg/kg/d endostatin microvessel density less than the saline control group, the difference was statistically significant (P = 0.006), while 2.5mg/kg/d and 10mg/kg / d group, with no significant difference between the saline group, P = 0.468 and 0.220, respectively. 5. Microvascular endothelial cell migration and reduce the number of endostatin in mice 16 hours after the intervention, the acting with endostatin role concentration increased (P = 0.000). The corresponding concentration intervention microvascular endothelial cell proliferative activity showed no statistical significance (P = 0.484). And inhibit migration corresponding endostatin intervention raised mice 16 hours after microvascular endothelial cell line forkhead box protein O1 of the expression levels of mRNA and total protein (P <0.05), and no change in the expression level of the phosphorylated proteins (P = 0. .943), to unphosphorylated forkhead box protein 01 expression based, and this effect is increased as the concentration increased endostatin. 7. Transplanted tumor tissue, cell suspensions by magnetic bead positive sorting before and after CD31-positive cells were 17.2% and 90.9%, vascular endothelial cells purified. 8.5mg/kg/d endostatin intervention 12 days after transplantation tumor tissue CD3 1 positive cells phosphorylation of forkhead box protein O1 expression levels and saline control group, the difference was not statistically significant (P = 0.896), total fork head box O1 protein expression is higher than that of the saline control group (P = 0.047), mainly to increased phosphorylation of forkhead box 01 protein expression; CD31 negative cells without and bead combined expression of the protein. Conclusion 1. Endostatin inhibit Namalwa lymphoma in nude mice transplanted tumor growth, tumor burden larger inhibitory effect remained significantly. 2. Endostatin play inhibitory effect requires the right dose, excessively high or low doses of endostatin antitumor effect of dampening. 3. Endostatin inhibit Namalwa lymphoma xenograft tumor growth and reduce tumor angiogenesis. 4. Endostatin mouse microvascular endothelial cell migration endostatin one of the ways to reduce tumor angiogenesis. 5. Endostatin to mice microvascular endothelial cell migration and raised unphosphorylated Foxo1 protein expression. 6. The endostatin raised unphosphorylated Foxo1 protein expression in vascular endothelial cells within the tumor tissue and reduce Namalwa lymphoma the transplanted tumor angiogenesis, unphosphorylated Foxo1 upregulated may be one of the mechanisms of the pharmacological effects of endostatin.