Effects Of Angiostatin On The Biological Behavior Of Lung Carcinoma And Its Inhibiting Mechanism Of Tumor Angiogenesis

Introduction and ObjectiveNeovascularization for unlimited malignant tumor growth and metastasis has important significance.Malignant solid tumors mainly composed of two parts,that is the tumor cells and stromal tumor.And the occurrence,development and transfer of tumor closely related to the growth of blood vessels in supportive stroma.When the tumor grew to 1-2 mm in diameter(containing about 106 cells),if there is no nutrition and oxygen supply vessels,the tumor cells will remain dormant or degradation occurred, and stop the proliferation or even death.With this theory,Folkman puts forward the hypothesis of "inhibit tumor angiogenesis" as a new ways of treatment for solid tumors. Over the past 20 years,the inhibition of tumor angiogenesis has become an important aspect of research in the control tumor growth,invasion and metastasis.Angiostatin is the first in recent years found that endogenous tumor angiogenesis inhibitor,which specifically affect directly vascular endothelial cells,blocking the proliferation and migration,sequentially reducing rumor angiogenesis.Although there are a lot of research reports about angiostatin at home and abroad,the mechanism of antiangiogenic action of angiostatin has not explained fully so far,the cell signaling pathway of angiostatin acting on the vascular endothelial cells has not been proven. Preliminary results of the study have revealed the mechanism angiostatin anti-tumor angiogenesis is a multi-factor.Angiostatin can inhibit proliferation,migration of endothelial cells,and induce apoptosis of endothelial cells to achieve anti-angiogenesis effect,thereby inhibiting tumor growth,invasion and metastasis.In order to accurately evaluate the action of anti-neovascularization and understand of anti-angiogenesis mechanism about angiostatin,we investigate the relationship between expression of angiostatin and tumor angiogenesis in the tumor tissue and the regulation of angiogenesis factor VEGF link in the body material,animal and in vitro cell culture experiments at three levels.And further definitude the impact of angiostatin on tumor growth and the major signaling pathway on angiostatin inhibiting proliferation of endothelial cell and apoptosis regulation,simultaneity explore the molecular mechanism of its role.These researches may provide some theoretical references and foundational understandings for the prevention and treatment of tumors.Materials and Methods1.Human materialsRT-PCR was used by detecting expression of VEGFmRNA and angiostatin mRNA in human lung carcinoma and relationship between its and Microvascular density,elucidating role of angiostatin in the process of occurrence and development of lung carcinoma and relationship between its and clinical pathology.2.Animal experimentsA₅₄₉ human lung carcinoma cells were used by establishing c57BL / 6 inbred mouse xenograft model experiments to evaluate the effectiveness of Angiostatin treatment of lung carcinoma.48 of human lung carcinoma A₅₄₉ C57BL / 6 inbred mice (subcutaneous xenografts)be divided into four groups,12 mice each groups,three groups are experiment,one is control.Subcutaneous injection Angiostatin0.2ml (concentration of 0.6 mg/ Kg,1.2 mg/ Kg,2.4 mg / kg) in the tumor of mice.Control group was injected with normal physiological brine equate.Injection number 1 / 2 d, and 14 days of treatment,tumor changes was observed.And using conventional pathological and immunohistochemical S-P methods were used by detecting of cellular morphological changes and CD34,Survivin and NF-κB expression in the tumor tissue, counting microvascular density(MVD),observating Angiostatin's effect on human lung carcinoma in mice transplanted tumor angiogenesis.3.Cells culture in VitroUsing primary cells cultured mathods culture human umbilical vein endothelial cells and A₅₄₉ human lung carcinoma cells in the study in vitro.Angiostatin impacting on proliferation activity of HUVECs and A₅₄₉ human lung carcinoma cells are examined by MTT colorimetric assay.Trypan blue stain cells counting was used observed angiostatin's influence on the growth of A₅₄₉ human lung carcinoma and vascular endothelial cells.Establishing of tumor microvascular model was used to observed angiostatin's role for endothelial cells shaping vascular cavity.With the flow cytometry,angiostatin on the impact on HUVECs cell cycle was analysed.Through western blot,we detect angiostatin imprinting of understandings on the role of vascular endothelial cell signal transduction pathway and its correlative factors,preliminary study the molecular mechanism of angiostatin anti-angiogenesis.Results1.Detection of human materials:Angiostatin mRNA in lung carcinoma tissue for the expression of 5.56±1.40, adjacent lung tissue for the expression of mRNA 7.85±1.74;VEGF mRNA expression in lung carcinoma tissues for 12.43±1.58,adjacent lung tissues to 9.10±1.20. Difference between groups was significant(P＜0.05);Angiostatin mRNA inâ… -â…¡Phase lung carcinoma tissue for the expression of 9.41±2.29,â…¢-â…£lung carcinoma tissue for the expression of 5.26±2.39,the latter than the former obvious expression level lower,the difference was significant(P＜0.05);metastasis group angiostatin-mRNA expression levels lower than those without metastasis group,the difference was significant(P＜0.05).VEGF mRNA expression inâ… -â…¡Phase lung carcinoma 9.79±1.45,expression inâ…¢-â…£lung carcinoma for 16.83±2.38,the latter expression levels were significantly higher than the former,the difference was extremely significant(P＜0.01);In cancer Section of the poorly differentiated cells and high - compared differentiation,there was a significant difference(P＜0.05),expression in the lymph node metastasis group was higher than that without lymph node metastasis group,the difference is extremely significant(P＜0.01).2.Animal experiments showed that:(1) Weights and inhibitory rate of tumorTumor cells inoculated with the increase in time,the tumors each groups of mice are gradually increasing,but the angiostatin treatment groups significantly slow tumor growth.Compared with the control group,three dose groups of angiostatin have anti-tumor effect averagly,the maximum inhibition of 77%(p＜0.05);(2) Microvessel dentisy of tumorCD34 expression:there all have expression in control group and tumor treatment groups.Under the microscope,CD34 diffuse distribution,in brown,yellow positive cells;Statistical analysis showed that tumor microvessel density of angiostatin treatment groups decreased,with dose angiostatin increasing MVD reduce.Compared with the treatment groups and control group,there were significant differences(p＜0.05).(3) Expression of survivin and NF-κBThe positive reaction products of survivin and NF-kB distribute in the nucleus and cytoplasm of the tumor cells,was brown.The positive rates in each group of survivin protein were as follows:control group,100%(12/12),angiostatin small dose group 91.6%(11/12),in the middle dose group 75%(9/12),66.6%of high-dose group(8/12); The positive rates of NF-kBp65 in the control group and angiostatin low dose group,is the same expression,for 33.3%(4/12),16.7%in the dose group(2/12),high-dose group was 25%(3/12);Image analysis showed the expression of NF-kBp65 in control group of vascular endothelial cell is 0.313±0.025,the expression of survivin in control group of vascular endothelial cell is 0.295±0.027.With Angiostatin dose increase,the expression of NF-kBp65 and Survivin in endothelial cell decreased gradually. Compared with the control group there is statistical significance(P＜0.05).3.experiments in vitro:(1) MTT assay results showed that:Different concentrations of angiostatin act in human umbilical vein endothelial cells after 24 h,OD₄₉₀ absorbance values were lower than the control group,its inhibiting action on the proliferation of endothelial cells with increasing concentrations of angiostatin increased,compared with the control group differences were significant (P＜0.01).And along with extension of acting time,the proliferation of human umbilical vein endothelial cells inhibited significantly,its absorbance values were lower than the control group.Measurement results show that:when 72h,the maximum inhibition rate reached 91.37 percent,compared with the control group the difference was significant(P＜0.01).(2) Flow test results of the cell cycle:Compared with different concentrations Angiostatin group and the control group, the proportion of cells in G1 phase has declined,in S phase has increased.From 4 mg/L angiostatin on,apoptosis began,and to 16 mg/L angiostatin,there is a clear peak of apoptosis,and increased with the dose,apoptosis peak is gradually strong.(3) Test of cavity formation in endothelial cells:After human umbilical vein endothelial cells were cultured in Tail plastic of mice and induced by tumor supernatant,endothelial cells of morphology have been observed from scattered in the state to gathered,gradually cord-like dendritic formation,12h reticular formation obvious.In cultured human umbilical vein endothelial cells after 3 h (vascular network has not formed before) by adding angiostatin,endothelial cells have not formed network structure.When endothelial cells were cultured for 24h(vascular network has been formed) by adding angiostatin,we discovered 8h endothelial cells decrease in the number of formating networks and became round,and gradually gathered,12h blood vessels network structure formed by endothelial cells have been ruptured,and disappear after fracture,the remnants of the endothelial cells form cells lumped together.(4) Flow detection expression of integrinαvβ3 in endothelial cells:FCM results showed,the average fluorescence density expression in control group is 6.76±2.19,pure bFGF group is 42.3±4.41,Angiostatin low dose group is 24.8±4.26,the high-dose group of angiostatin is 2.34±1.16.Comparing groups,there is a relatively obvious statistical significance.This results indicate that angiostatin restrained the expression of integrinαvβ3 in endothelial cells by bFGF induction.(5) Western Blot detection Angiostatin role after the angiogenesis pathwayAfter reaction of endothelial cells and spicific signal pathway inhibitors Akt,P38, FAK protein expression levels are relatively low,bFGF stimulated slightly higher.At the same time in the endothelial cells after treated with Angiostatin,expression levels of NF-κBp65 and Survivin increased with the dose of Angiostatin reduced.Conclusions1.The expression of Angiostatin in human non-small cell lung carcinoma correlate with the clinical tumor stage,differentiation and lymph node metastasis.2.Angiostatin can restrain the growth on A₅₄₉ lung carcinoma in mice transplanted tumor and inhibit neovascularization.3.Angiostatin can inhibit the proliferation of human vascular endothelial cell incubated in vitro and induce its apoptosis,and restrain blood vessel cavity formation induced by tumor in vitro.4.Angiostatin related with inhibition of endothelial cell proliferation or induction of apoptosis and the role of the integrin avβ3 expression and Survivin,NF-κB.