The Effect And Mechanism Of Bevacizumab And E109 On Biological Behavior Of A549 Cells Under Either Hypoxia Or Hypoxia Combined With Serum Starvation Condition

ObjectiveTo observe the effect of antiangiogenic dr?g bevacizumab and small molecule tyrosine kinase inhibitor E109 on the biological behavior of A549 cells,involving in cell proliferation,invasion,migration,under hypoxia or hypoxia combined with serum starvation condition.And by high-thro?ghput sequencing to explore the‘activated genes'which change the biological behavior of A549 cells induced by high concentrate of bevacizumab under hypoxia or hypoxia combined with serum starvation condition.Meanwhile compare the changes of the‘activated genes'in A549 cell treated with E109 to search the methods of overcoming resistance to angiogenesis.Method:The vitro hypoxia model was established by the cell hypoxia apparatus of Billups-rothenberg,which was full of the mixture gas?1%O2+5%CO2+94%N₂?.MTT was adopoted to detect the proliferation ability of A549 cell under hypoxia or hypoxia combined with serum starvation condition.Invation and migration ability of A549 cell was observed by transwell invation and migration assay.Flow cytometry was used to detect the changes of cell cycle.By high-thro?ghput sequencing to explore the differential gene which change the biological behavior of A549 cells induced by high concentrate of bevacizumab under hypoxia or hypoxia combined with serum starvation condition.Then the differential gene was verificated by real-time PCR technique.The expression of RGC32,E-cadherin,Snail+Sl?g,MMP-2were detected by western-blot.Results:1.The results of MTT methods:?1?After bevacizumab increased to a certain concentration,the inhibition rate of A549 cell was also raised as the increased concentrate of bevacizumab under hypoxia condition.However the difference was not statistically significant.Under hypoxia combined with serum starvation condition,after bevacizumab increased to a certain concentration,the inhibition rate of A549 cell was also raised as the increased concentrate of bevacizumab. And the difference was statistically significant.?2?As the increased concentrate of E109 treated with A549 cell,the inhibition rate of A549 cell was raised under both hypoxia and hypoxia combined with serum serum starvation condition.And the difference was statistically significant.2.The results of cell cycle:Compared with the control group,the group of Bev80?g/ml and Bev 160?g/ml after treated with A549 cell 24h,the proportion of G0/G1 phase was decreased and the proportion of S phase was increased under both hypoxia and hypoxia combined with serum serum starvation condition.. However the difference was not statistically significant.3.The results of Transwell experiment:?1?Compared with the control group,the group of Bev80?g/ml and Bev 160?g/ml after treated with A549 cell,the ability of invation and migration was increased under both hypoxia and hypoxia combined with serum serum starvation condition.And the difference was statistically significant.?2?The migration ability of A549 cell was decreased as the concentrate of E109 was increased and the difference was statistically significant.4.The results of high-thro?ghput sequencing:Compared with the control group,the group of Bev80?g/ml and Bev160?g/ml after acted on A549 cell,the expression of RGC32 gene was upregulated under hypoxia or hypoxia combined with serum starvation condition and the difference was statistically significant.5.The results of real-time PCR:?1?Compared with the control group and Bev20?g/ml group,the group of Bev80?g/ml and Bev160?g/ml after acted on A549 cell,the relative RGC32 mRNA expression was upregulated under hypoxia or hypoxia combined with serum starvation condition and the difference was statistically significant.?2?The relative RGC32 mRNA expression was decreased as the concentrate of E109 was increased under hypoxia or hypoxia combined with serum starvation condition and the difference was statistically significant.6.The results of western blot:?1?Compared with the control group,the Bev80?g/ml and Bev160?g/ml group can significantly up-regulate the level of protein expression of RGC32 and EMT related makers such as Snail+Sl?g and MMP-2 and can down-regulate the level of protein expression of E-cadherin.?2?The level of RGC32 MMP-2 and Snail+Sl?g protein expression was decreased and the level of E-cadherin protein expression was up-regulated as the concentrate of E109 was increased under hypoxia or hypoxia combined with serum starvation condition and the difference was statistically significant.Conclusion:1.High concentration of bevacizumab can promote the invasion and migration ability of A549 cell which means the occurrence of malignant biological behavior of A549 cell.2.As the increased concentrate of E109 treated with A549 cell,the cell proliferation rate and the ability of migration was decreased under both hypoxia and hypoxia combined with serum starvation condition.3.High concentration of bevacizumab can up-regulate the expression of RGC32 both in mRNA and protein level to further modulate related makers of EMT which eventually promotes the occurrence of malignant biological behavior of A549 cell under hypoxia or hypoxia combined with serum starvation condition.