Propofol Combined With Ulinastatin Suppress Human Lung Adenocarcinma A549 Cell Migration And Invasion

ObjectiveThe migration and invasion of tumor are the most important steps in tumor metastasis.Both propofol and ulinastatin can inhibit the migration and invasion of tumor.The aim of this study is to evaluate the synergistic effects of propofol combined with ulinastatin and different drug administration schedules using propofol and ulinastatin against the migration and invasion of human lung adenocarcinoma A549 cells,in order to explore the best administration sequence of propofol and ulinastatin.At the same time by clarifying the expression of E-cadherin in lung adenocarcinoma A549 cells to study the mechanism of propfol combined with ulinastatin suppress human lung adenocarcinoma A549 cell migration and invasion,which provides a theoretical basis for the reasonable application of propofol and ulinastatin in lung cancer operation.Method1.Cell preparation and groupingThe A549 cells of human lung adenocarcinoma from the logarithmic growth period were divided into 7 groups:(1)Group control:continues treatment with the serum-free medium for 48 hours(Control);(2)Group DMSO:continues treatment with 0.1%DMSO for 48 hours(DMSO);(3)Group ulinastatin:continues treatment with 200 U/mL ulinastatin for 48 hours(UTI);(4)Group propofol:continues treatment with serum-free medium for 42 hours,followed by 30μM propofol for 6 hours(PPF);(5)Group propofol combined with ulinastatin:continues treatment with 200U/mL ulinastatin for 42 hours,followed by 30μM propofol and 200U/mL ulinastatin for 6 hours(UTI+ PPF).(6)Group propofol→ulinastatin:continues treatment with 30μM propofol for 6 hours,followed by 200U/mL ulinastatin for 42 hour(PPF→UTI);(7)Group ulinastatin→propofol:continues treatment with 200 U/mL ulinastatin for 42 hours followed by 30μM propofol for 6 hours(UTI→PPF).2.Transwell migration essayWhen the 7 groups of cells were fused to 80%-90%growth,starving the cell with the serum-free medium for 12 hours,then receiving either group treatment for 48 hours.After digesting and centrifuging,the cells were resuspended to 106cells/ml and seeded in the upper chamber(100 μL),while the DMEMF/F-12 with 10%fetal bovine serum was joined in the lower chamber(600pL).After cultured for 24 hours take out the upper chamber,wed swab remove the cells on the upper membrane without migration.Cells were fixed with anhydrous methanol for 20 minutes and stained with 0.1%crystal violet for 15 minutes.Regularly select 9 microscope visual fields and photograph.Count the cell number and averaged.The inhibition rate of cell migration/%=(1-the migrating cell number of experimental group/the migrating cell number of control group)*100%.3.Transwell invasion essayPlate the Matrigel subsrate(100μL)on the PET membrane of each upper chamber in advance.The residual operation was carried out with the Transwell migration essay.The inhibition rate of cell invasion/%=(1-the invasion cell number of experimental group/the invasion cell number of control group)*100%.4.Western blotDetect the expression of E-cadherin at protein level in each group cells by Western blot.5.Statistical analysisData were expressed as the means ± standard(SD)of three replicates.The principal statistical test was the one-way analysis of variance(ANOVA).After determination of variance by F-test,the Dunnett’T3 were to assess the statistical significance,with statistical significance defined as follows:P<0.05.Result1.Transwell migration essay assessment of migration of A549 cells Compared with Control,the A549 cell migration ability of PPF,UTI+PPF,PPF→UTI and UTI→PPF was significantly decreased,and the cell migration inhibition rates were 41.91%,63.97%,65.45%and 65.07%,respectively,and the difference was statistically significant(P<0.05).The A549 cell migration ability of UTI weaker than that of Control,but the difference was not statistically significant.Compared with PPF,the migration ability of A549 cells in UTI+PPF,PPF→UTI and UTI→PPF decreased more obviously,and the difference was statistically significant(P<0.05).There was no significant difference in migration ability of A549 cells in UTI+PPF,PPF→UTI and UTI→PPF.There was no statistical difference between Control and DMSO.2.Transwell invasion essay assessment of invasion of A549 cellsCompared with Control,the A549 cell invasion ability of PPF,UTI+PPF,PPF→UTI and UTI→PPF was significantly decreased,and the cell invasion inhibition rates were 34.81%,65.99%,67.61%and 61.95%,respectively,and the difference was statistically significant(P<0.05).The A549 cell invasion ability of UTI weaker than that of Control,but the difference was not statistically significant.Compared with PPF,the invasion ability of A549 cells in UTI+PPF,PPF→UTI and UTI→PPF decreased more obviously,and the difference was statistically significant(P<0.05).There was no significant difference in invasion ability of A549 cells in UTI+PPF,PPF→UTI and UTI→PPF.There was no statistical difference between Control and DMSO.3.Detection of the expression of E-cadherin by Western blotCompared with Control,the expression of E-cadherin protein in PPF,UTI+PPF,PPF→UTI and UTI→PPF increased,and the difference was statistically significant(P<0.05).Compared with PPF,the expression of E-cadherin protein in group UTI+PPF,PPF→UTI and UTI→PPF increased more obviously,and the difference was statistically significant(P<0.05).There was no significant difference in the expression of E-cadherin protein in group UTI+PPF,PPF→UTI and UTI→PPF.There was no statistical difference between Control,DMSO and UTI.Conclusion1.Propofol can inhibit the migration and invasion of A549 cells,and the combination of propofol and ulinastatin is more effective with the clinical concentration.However,the clinical concentration of ulinastatin has no significant inhibitory effect on the migration and invasion of A549 cells.2.There is no significant difference in the inhibition of migration and invasion of A549 cells in different schedules and timing of propofol and ulinastatin with the clinical concentration.3.With the clinical concentration,propofol and ulinastatin inhibit the migration and invasion of A549 cells,which may be related to the promotion of E-cadherin protein expression and the inhibition of EMT in A549 cells.