The Effect Of Antiangiogenic Agents On Abilities Of Invasion And Metastasis Under Serum Starvation And Hypoxia In Human Breast Carcinoma Cells

ObjectivesTo observe the abilities of invasion and metastasis under serum starvation of MDA-MB-231 cells under serum starvation and hypoxia;and the effect of antiangiogenic drugs,rh-endostatin and bevacizumab on the abilities of invasion and metastasis under serum starvation and hypoxia in breast carcinoma cell.Investigate the potential risk of long term antiangiogenic therapy in clinical application.MethodCCK-8 was conducted for detecting the working concentration and time of rh-endostatin and bevacizumab as cell inhibition rate was about 30%.Cells were randomized into 12 groups,group a:al,10%fetal bovine serum(FBS)control group;a2,10%FBS+ rh-endostatin group;a3,10%FBS+ bevacizumab group;group b:b1,hypoxia +10%FBS control group;b2,hypoxia+10%FBS+ rh-endostatin group,b3,hypoxia+ bevacizumab group;group c:c1,serum starvation control group;c2,serum starvation + rh-endostatin group;c3,serum starvation + bevacizumab group;group d:dl,hypoxia+ serum starvation control group;d2,hypoxia+ serum starvation+rh-endostatin group;d3,hypoxia+ serum starvation+ bevacizumab group.Cells were detected in vitro invasive and migration transwell experiments.The expression of c-Met and MMP-9 were detected by western blot.Cells treated with rh-endostatin or bevacizumab under serum starvation and serum starvation control group were detected by chip hybridization with miBase 18.0 microarray.QRT-PCR assay was conducted for detecting the level of 10 dysregutated microRNAs,miR124,miR505,miR2355,miR10a,miR576,miR1915,miR375,miRlet-7d,miR519,miR211,which are correlated with oncogenic or tumor suppressing functions.Results1.The results of CCK-8 experiment:The inhibition rate of MDA-MB-231 cells was about 30%when the concentration of rh-endostatin was 800mg/L and bevacizumab was 80mg/L during 48 hours.We considered 30%as valid inhibition and choosed 800mg/L of rh-endostatin and 80mg/L of bevacizumab as working concentration and 48 hours as working time.2.The results of invasive and migration transwell experiment:Cells of serum starvation group penetrated more cells than 10%FBS control group.MDA-MB-231 cells were not vulnerable to hypoxia.Cells treated rh-endostatin/bevacizumab group under serum starvation penetrated the matrigel and basilar membrane were much more than the serum starvation control group(P<0.05).3.The results of Western Blot:under serum starvation the expression of c-Met and MMP-9 were more than the 10%FBS control group.After treatment of rh-endostatin/bevacizumab under serum starvation environment the expression of c-Met and MMP-9 were increased significantly compared with serum starvation control group(P<0.05).4.The results of miRNA microarray expression analysis:The results presented as fold change(FC).There are 10 oncogenic-related miRNAs among 38 dysregulated expression miRNAs.They are miR-124,miR-505,miR-519,miR-211,miR-10a,miR-375,miR-1915,miR-2355,miR-let-7d,miR-576.5.The results of Real-time PCR:The level of miR-2355 and miR375 were decreased inserum starvation group,in rh-endostatin/bevacizumab group under serum starvation group the level of miR-2355 and miR375 were much lower than the serum starvation group.(P<0.05).ConclusionWe simulated the environment after potent antiangiogenic therapy in vitro,found that serum starvation increased the abilities of invasion and metastasis of MDA-MB-231 cells.Antiangiogenic drugs increased the abilities of invasion and metastasis under serum starvation environment in breast carcinoma cells from cellular,protein and genetic level.MDA-MB-231 cells were not vulnerable to hypoxia.MiR375 and miR2355 may be proposed to be early warning markers to determine whether excessive antiangiogenic therapy exists.