Effects About ATRA On Proliferation And The Expression Of COX-2 Of Human Lung Adenocarcinoma Cell Line A549

Abstract:Objective:To investigate the possible effects and mechanisms about all-trans retinoic acid (ATRA) on proliferation and the expression of COX-2 of lung adenocarcinoma cell line A549 by research the effects about ATRA of cells in vitro. Methods:(1) Cultivating of A549 cells:Inoculate A549 cell line in 25ml culture bottle, and cultivate them by RPMI1640 medium which containing 10% FBS, in 5%CO2,37℃incubator. (2) A549 cells of logarithmic period were used in the experiment. There were divided into three time periods by the time that the A549 cells were treated with ATRA after 24 hours,48 hours and 72 hours. Each time period was divided into 5 groups:drug concentration of 1×10-7mol/L(Group2), 1×10-6mol/L(Group3), 1×10-5mol/L (group4), 1×10-4mol/L(Group5), and 1 control group(Group1)that without any drug. There are 10 holes in each group. MTT was detected in all groups after adding ATRA 24 hours,48 hours and 72 hours. Calculate the cell growth inhibition rate. (3) Select the appropriate time and different drug concentration and detect the expression of cyclooxygenase-2 (COX-2) in the cytoplasm of these cells by the way of immunocytochemical SP(streptavidin-peroxidase). Cultivate the cells without drug but in the same conditions as the control group, compare the expression of COX-2 between the experimental groups and the control group. (4) Select the appropriate time and different drug concentration and detect the expression of cyclooxygenase-2 (COX-2) in the supernatant preparations of these cells by the way of enzyme linked immunosorbent assay (ELISA). Cultivate the cells without drug but in the same conditions as the control group, Compare the expression of COX-2 between the experimental groups and the control group. (5) Select the appropriate time and different drug concentration and detect the cell apoptotic alterations of these cells by the way of transmission electron microscope. Cultivate the cells without drug but in the same conditions as the control group, Compare apoptotic alterations of the cell between the experimental groups and the control group. Results:(1) After ATRA effects A549 cell for 24 hours, there was no difference in the IOD of cells between the control group and experimental groups which concentrations of 1×10-7mol/L and 1×10-6mol/L(P>0.05). The results show that the ATRA with these drug concentrations can not inhibit the proliferation of A549 cells. The difference was statistically significant when the concentration of 1×10-5mol/L and 1×10-4mol/L compared with the control group respectively(P<0.05). This indicates that the ATRA with these drug concentrations can inhibit the proliferation of cells. There are no significant differences between the adjacent experimental groups(P>0.05). With different concentrations of ATRA affecting lung adenocarcinoma A549 cells for 24 hours, the proliferation of cells can be inhibited when the ATRA reached a certain concentration. (2) After ATRA effects A549 cell for 48 hours, there was no difference in the IOD of cells between the control group and experimental groups which concentrations of 1×10-7mol/L(P>0.05). The result shows that the ATRA with this drug concentrations can not inhibit the proliferation of cells. When the experimental groups which concentration of 1×10-6mol/L, 1×10-5mol/L and 1×10-4mol/L compared with the control group respectively, the results show that the ATRA with these drug concentrations can inhibit the proliferation of cells. There are significant differences between the adjacent experimental groups(P<0.05). With different concentrations of ATRA group affecting lung adenocarcinoma A549 cells for 48 hours, the proliferation of cells can be inhibited when the ATRA reached a certain concentration. And with the drug concentrations rising the inhibition become more obviously. (3) After ATRA effects A549 cell for 72 hours, the difference was statistically significant when the experimental groups which concentration of 1×10-7mol/L, 1×10-6mol/L, 1×10-5 mol/L and 1×10-4mol/L compared with the control group respectively (P<0.05). The results show that the ATRA with every experimental drug concentration can inhibit the proliferation of cells. There are significant differences between the adjacent experimental groups(P<0.05). With every experimental concentration of ATRA affecting lung adenocarcinoma A549 cells for 72 hours, the proliferation of cells can be inhibited. And with the drug concentrations rising the inhibition become more obviously. (4) The expression of COX-2 in the cytoplasm decreased after the A549'cells were treated by ATRA with different concentration for 72 hours. It Shows that there is significant inhibition to the cells in this effect conditions compared with the control group(P<0.05), And indicates that COX-2 maybe participate in the process of inhibition the proliferation of the cells. (5) The expression of COX-2 in the Supernatant preparations decreased after the A549 cells were treated by ATRA with different experimental drug concentration for 72 hours. It Shows that there is significant inhibition to the cells in this effect conditions compared with the control group(P<0.05), And indicates that COX-2 maybe participate in the process of inhibit the proliferation of cells. (6) Typical features of apoptosis was found by transmission electron microscope after the A549 cells were treated by ATRA with different concentrations for 72 hours. It Shows that there is significant inhibition to the cells in this effect conditions compared with the control group(P<0.05), And indicates that ATRA can inhibit the proliferation of A549. (7) Conclusion:ATRA inhibit the proliferation of lung adenocarcinoma A549 cells might through decreasing the expression of COX-2. This observation may provide new evidence for ATRA in chemoprevention of lung cancer.