Effects Of Rotundic Acid On Proliferation,migration And Invasion Ability Of Human Lung Adenocarcinoma Cells

Objective The purpose of this study was to investigate the effect of Rotundic acid on proliferation,migration and invasion ability of human lung adenocarcinoma cells as well as its possible mechanisms.Methods With human lung adenocarcinoma A549 and PC9 cells as research objects,the experiments were grouped as Control,including Blank Control Group（empty）,Solvent Control Group（containing 0.1%DMSO）and Cell Control Group（without RA or DMSO）,and Experimental Groups（containing 20,40,60 and 80 μmol/L RA,respectively）.Cells in logarithmic phase were selected and treated for 24 hours.CCK-8 assay was then used to detect the proliferation of A549 and PC9 cells in each group.Using scratch test,the horizontal migration ability of A549 and PC9 cells was detected;and the longitudinal migration ability of A549 and PC9 cells in each group was detected by Transwell migration assay.With Transwell invasion assay,the invasion ability of A549 and PC9 cells in each group was detected.In addition,The expression levels of Janus Kinase 2（JAK2）And Signal And Activator Of Transcription 3（STAT3）in A549 proteins in the supernatants of A549 and PC9 cells were detected by ELISA.The expression levels of JAK2 and STAT3 m RNA in the A549 and PC9 cells were detected by RT-PCR.Statistical analysis was then performed for the differences between groups in each index of experiment.Results（1）CCK-8 assay: There was the phenomenon of proliferation inhibition of different degrees in A549 and PC9 cells in the experimental groups compared with the cell control group.The higher the concentration of RA,the more obvious the inhibition of RA on cell proliferation.Compared with the control group,the solvent had no obvious inhibition on the proliferation of A549 and PC9 cells.The difference between the experimental groups and the cell control group was statistically significant（A549: F=103.1,P<0.05;PC9: F=121.8,P<0.05）.（2）Cell scratch test:The results indicated that,RA inhibited the horizontal migration of A549 and PC9 cells.The higher the concentration of RA,the smaller the migration area of human lung adenocarcinoma cells in the experimental groups.There were statistically significant differences between the experimental groups and the cell control group（A549: F=145.5,P<0.05;PC9: F=157.2,P<0.05）.（3）Transwell migration experiment: The phenomenon that RA inhibited the longitudinal migration of A549 and PC9 cells existed.When RA concentration increased,the number of cells crossing the polycarbonate membrane decreased.The difference of cell numbers between experimental groups and control was statistically significant（A549: F=610.9,P<0.05;PC9:F=317.1,P<0.05）.（4）Transwell invasion assay: The results showed that,the invasion ability of A549 and PC9 cells was decreased after RA treatment.The higher the RA concentration,the stronger the inhibition effect.The difference between the experimental groups and the control group was statistically significant（A549:F=320.3,P<0.05;PC9: F=246.1,P<0.05）.（5）ELISA: The results showed that,the higher RA concentration,the lower JAK2 and STAT3 protein expression in the supernatant of A549 and PC9 cells.There was statistical difference in the levels of JAK2 and STAT3 proteins in the supernatant of A549 and PC9 cells between the experimental groups and the control one [A549:（JAK2: F=21.29,P<0.05;STAT3:F=22.16,P<0.05）;PC9:（JAK2: F=22.73,P<0.05;STAT3: F=21.08,P<0.05）].（6）RT-PCR: It indicated that,the higher RA concentration,the lower the JAK2 and STAT3 m RNA expression levels in the cells of the experimental groups.The difference of JAK2 and STAT3 m RNA expression levels in A549 and PC9 cells between the experimental groups and the control one was statistically significant[A549:（JAK2: F=40.44,P<0.05;STAT3: F=32.04,P<0.05）;PC9:（JAK2: F=117.9,P<0.05;STAT3: F=129.0,P<0.05）].Conclusion RA may in vitro inhibit proliferation,migration and invasion of human lung adenocarcinoma A549 and PC9 cells in a concentration-dependent manner.The potential mechanisms of these effects might include negative regulation of the JAK2/STAT3 pathway in cells.