| Objective:1.The effect of antiangiogenesis drug bevacizumab on cell proliferation,migration,invasion and cell cycle of human lung adenocarcinoma A549 cell under nutritional condition and serum starved condition were observed.The expression level of related genes and proteins were clarified as well in order to explore the possible reason and related mechanism for the change of biological behavior of A549 cells caused by bevacizumab under nutritional condition and serum starved condition.2.By observing the effect of the new type multi-targeted drug E109 on cell proliferation,migration and expression level of related genes and proteins of A549 cells under nutritional condition and serum starved condition,we aimed to reveal the influence of E109 on biological behavior of A549 and explore its molecular mechanism.Methods:The concentrations of bevacizumab in the subsequent experiments were 40 ?g/ml,160 ?g/ml,240 ?g/ml,which were selected according to our preliminary Transwell experimental results.A549 cells with good condition were used in the expriments.MTT assay was used to detect the influence of bevacizumab on proliferation ability of A549 cells under nutritional condition and serum starved condition.The effect of bevacizumab on the migration and invasion ability of A549 was respectively detected by Transwell migration assay and Transwell invasion assay.Flow cytometry was applied to assess A549 cell cycle distribution.The sreening of realted differential gene expression was performed by next gene sequencing technology and the gene expression of RGC32(response gene to complement 32,also known as RGCC)was validated by RT-PCR methods.Western blot methods was used to observe the expression level of RGC32 protein,VEGF-A protein,MMP-2 protein and N-cadherin protein in A549 cells after processed by bevacizumab under nutritional condition.The expression level of RGC32 protein and MMP-2 protein in A549 cells which were handled with bevacizumab as well as PDTC(a kind of NF-?B inhibitor)were detected by Western blot methods.The effect of E109 on proliferation ability of A549 cells under nutritional condition and serum starved condition was detected by MTT assay.We chose the concentrations of which the cell growth inhibition rates are 5%(IC5),30%(IC30)and 50%(IC50)to observe the influence of E109 on A549 migration ability with Transwell migration assay.The gene expression of RGC32 was validated by RT-PCR methods.The expression level of RGC32 protein?MMP-2 protein and N-cadherin protein expression in A549 cells after processed by E109 under nutritional condition was observed by Western blot methods.Results:1.(1)After treatment for 24 h under nutritional condition,the A549 cell growth inhibition rates of Bev 40 ?g/ml,Bev 160 ?g/ml and Bev 240 ?g/ml were 13.22%,10.91% and 19.89% respectively,-10.18%,6.39% and-0.93% respectively for 48 h under nutritional condition,5.08%,24.19% and 25.33% respectively for 24 h under serum starved condition,6.79%,12.33% and 25.04% respectively for 48 h under serum starved condition.(2)A549 cells were processed with E109 of different doses under nutritional condition or serum starved condition.Under nutritional condition or serum starved condition,the inhibition rate increased gradually as the concentration of E109 elevated in a certain time(24h,48 h or 72h).The inhibition rate also increased gradually as the time prolonged in a certain concentration(1 ?mol/ml,2 ?mol/ml,4 ?mol/ml,8 ?mol/ml,12 ?mol/ml,16 ?mol/ml).2.(1)Under nutritional condition and serum starved condition,higher concentrations of bevacizumab(Bev 160 ?g/ml or Bev 240 ?g/ml)promoted the migration ability of A549 cells,compared with the control(both P<0.05),and lower concentration of bevacizumab(Bev 40 ?g/ml)inhibited the migration ability of A549 cells(P<0.05).(2)After treatment for 48 h under nutritional condition,A549 cell migration number of E109 2?mol/ml group,E109 8?mol/ml group and E109 16?mol/ml group reduced significantly compared with control group(all P<0.05).A549 cell migration number of E109 2?mol/ml group did not reduced obviously(P>0.05),while migration number of E109 8?mol/ml group and E109 16?mol/ml group reduced significantly(both P<0.05)after treatment for 24 h under nutritional condition,24 h under serum starved condition and 48 h under serum starved condition.3.Under nutritional condition and serum starved condition,lower concentration of bevacizumab(Bev 40?g/ml)inhibited A549 cells invasive ability(P<0.05)and higher concentrations of bevacizumab(Bev 160?g/ml and Bev 240?g/ml)promoted the invasive ability of A549 cells significantly(both P<0.05).4.According to the results of next generation sequencing,we reviewed existing literatures and pubmed,finding that expression of RGC32 gene which was related to cell biological behavior in Bev 160?g/ml group and Bev 240?g/ml group under nutritional condition were increased.5.After treatment for 24 h or 48 h under nutritional condition,cells of G0/G1 phase,S phase and G2/M phase of Bev 160 ?g/ml group or Bev 240 ?g/ml group had no difference with that of control group(all P>0.05).The cell cycle distribution did not change after treatment for 24 h under serum starved condition as well(P>0.05).After treatment for 48 h under serum starved condition,cell cycle distribution of Bev 160?g/ml group made no diference(P>0.05);cell number of G0/G1 phase increased in Bev 240?g/ml group(P=0.012),but cell number of G2/M phase and S phase did not change(P>0.05).6.(1)The lower concentration of bevacizumab treatment(Bev 40?g/ml)had no significant effect on RGC32 RNA expression under nutritional condition by RT-PCR(P=0.638),while higher concentration group(Bev 160?g/ml and Bev 240?g/ml)upregulated RNA expression of RGC32(P<0.001 and P=0.013 respectively).(2)RGC32 RNA expression of E109 2?mol/ml group,E109 8?mol/ml group and E109 16?mol/ml group showed no difference with control group under nutritional condition(P>0.05).7.(1)Under nutritional condition,lower concentration of bevacizumab treatment(Bev 40?g/ml)did not affect the expression of VEGF-A protein(P=0.796);although the expression of RGC32 protein,MMP-2 protein and N-cadherin protein decreased,there were no statistical significance with control group(P=0.070,P=0.291 and P=0.185 respectively).Bevacizumab of higher concentration(Bev 160?g/ml and Bev 240?g/ml)could upregulate the expression of RGC32 protein,VEGF-A protein,MMP-2 protein and N-cadherin protein significantly(P<0.05).(2)After treatment for 24 h under nutritional condition,RGC32 protein expression of E109 2?mol/ml group,E109 8?mol/ml group and E109 16?mol/ml group were downregulated(P<0.001);MMP-2 protein expression in E109 2?mol/ml group,E109 8?mol/ml group were increased,and decreased in E109 16?mol/ml group(P<0.001);expression of N-cadherin protein were upregulated in E109 2?mol/ml group,and downregulated in E109 8?mol/ml group and E109 16?mol/ml group(P<0.001).8.The expression of RGCC protein and MMP-2 protein in A549 cell were downregulated in the group with both PDTC and bevacizumab processed than that of the group treated with bevacizumab only.Conclusions:1.Under nutritional condition and serum starved condition,E109 could inhibit the proliferation of A549 cells,and the inhibitory effect showed a certain concentration and time dependent manner.The inhibitory effect of bevacizumab on proliferation of A549 cells did not depend on concentration or time.2.Under nutritional condition and serum starved condition,bevacizumab of higher concentration could promote the migratory and invasive ability of A549 cells.Higher concentration of bevacizumab could also induce A549 cell EMT which may be caused by VEGF-VEGFR autocrine loop or by upregulation of RGC32 or the result of their interaction.NF-?B inhibitor PDTC may impair the EMT change caused by bevacizumab.However,the specific mechanisms of EMT induced by bevacizumab should be further validaed.3.Under nutritional condition and serum starved condition,E109 could suppress the migratory ability of A549 cells.Elevated concentration of E109 could inhibit A549 cell EMT under nutritional condition,however,further explorations are needed for the specific mechanisms. |
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