The Effects Of 4-amino-2-trifluoromethyl-phenyl Retinate On The Proliferation And Apoptosis Of Human Lung Adenocarcinoma A549 Cell Lines And Its Possible Mechanisms

Background: Lung cancer is one of the malignant tumors to theaten the mankind life healty, which is the leading cause of all cancer death Due to the growing morbidity and mortality of lung cancer patients, it is extremely urgent. to explore a novel effectively therapeutic method. Objective: To investigate the effects of a novel all-trans retinoid acid(ATRA) derivative, N-(3-trifluoromethyl-phenyl)-retinamide(ATPR) on the proliferation and apoptosis of lung adenocarcinoma A549 cells in vitro and to explore its probable mechanism. Methods: A549 cells were treated with ATPR and ATRA for 48 h. MTT and plate colony formation assay were used to assess the cytotoxicity of ATPR and ATRA on A549 cells. The expression levels of proteins related to proliferation(including ASPP family, WP53, P21, MDM2) and apoptois(including BCL-2, Bax, pro-caspase3, NF-κB(P65)were analyzed by western blot. Hoechst staining was performed to observe the effect of ATPR on the apoptosis of A549 cells. Results: for MTT assay, after A549 cells were treated with different concentrations of ATPR or ATRA(5, 10, 20, 25, 50, 75, 100 μmol/L) for 48 h, then incubated with MTT for 4 h. the relative viability rate which was converted from absorbancy value of A549 cells was markedly reduced in ATPR group(from 50 μmol/L to 100 μmol/L) in a dose-dependent manner but had little change in ATRA group compared with control group. According the above data, 50 μmol/L was chosen as the treatment concentration for the following assays. For plate colony formation assay, signal A549 cells which had been treated with 50 μmol/L ATPR or ATRA were then incubated for 8-10 days. Then A549 cells were treated with crystal violet. The observation of naked eyes and microscope showed that colony numbers and cell numbers of each colony were markedly reduced in treatment groups compared with control group. Moreover, the reduction level of ATPR group was more notable than that of ATRA group. After A549 cells were treated with ATPR and ATRA for 48 h, the expression levels of ASPP family, WP53, MDM2 and P21 proteins of each group cells were detected by Western blot. And compared with control group, the grey value of iASPP, P21 was decreased, while of WP53, MDM2 was increased, but of ASPP1,ASPP2 had no definite change. Moreover, the influences of ATPR on above related proteins expression were remarkable than that of ATRA. For Hoechst staining, the apoptosis of A549 cells treated with either ATPR or ATRA was observed with inverted fluorescence microscope which made the cell numbers significantly reduce, with concentrated chromatin and the appearance of apoptosis bodies. However, the number of apoptosis bodies of ATPR group was more than that of ATRA group. The expression levels of BCL-2, Bax, pro-caspase3 and NF-κB(P65) were detected by Western blot and compared with control group, the grey value of BCL-2, pro-caspase3 and NF-κB(P65) was decreased, while that of Bax was increased. Moreover, the influences of ATPR on above related proteins expression were remarkable than that of ATRA. Conclusion: ATPR inhibited A549 cells proliferation by downregulating the expression of iASPP and enhancing the WP53-mediating decreasing of BCL-2/Bax to activate apoptosis.