The Role Of INOS In EGCG Promoting A549 Cell Apoptosis

ObjectivesTo investigate the effect of inducible NOS(iNOS) in EGCG promoting apoptosis of A549cells.Materials and Methods1. MaterialsHuman lung adenocarcinoma A549cells (Medical Center of Affiliated Hospital of Qingdao University, Medical College)RPMI1640and fetal bovine serum (Hyclone)Ribbit anti human iNOS polyclone antibody (Abcam)Annexin V-FITC/PI Apoptosis Assay System (BD, America)Steady-Glo(?) Luciferase Assay System (Promega, America)2. MethodsThe effect of EGCG on the proliferation of A549cells were detected by MTT assay. A549cells were cultured in vitro, the cells in logarithmic growth phase were selected and divided into2groups:a control group and EGCG groups which administrated with normal saline and EGCG at the concentration of50mg/L-400mg/L respectively for24hours. The MTT (5mg/ml,20μl) were applified and the light absorption value were detected using an ELISA plate reader at570nm. All experiments were performed in quintuplicate. The cell viability was calculated with Karber. The apoptosis effect of EGCG in A549cells by flow cytometry. The A549cells in logarithmic growth phase at3x105cells/well were seeded in6-well plates and cultured overnight. The80-90%confluent A549cells were starved for4h in serum-free RPMI1640medium before treated with normal saline and EGCG at the concentration200mg/L respectively for24h at37℃, then the cells were collected and stained with PE and7-AAD and examined by flow cytometry with Beckman Coulter CyAn9-color high-speed analyzer. The expression of iNOS, proteins as well as their mRNA, was detected with Immunocytochemical staining, western blotting and Real time-PCR analyses respectively.3. ResultsThe results, compared with that in control group, showed that EGCG significantly inhibited proliferating activity of A549cells at the concentration of50mg/L-400mg/L (F=1000.35, q=3.43～60.00, P<0.05). The role of EGCG at the concentration of200mg/L was the most significant. EGCG at200mg/L advanced early and late apoptosis rate in A549cells, especially the late apoptosis with significant difference (x2=65.23, P<0.05). The small cells with cytoplasm and nucleus pyknosis caused by EGCG were observed. EGCG reduced the expression of iNOS protein and mRNA in A549cells, with significant difference (t=9.4, p<0.05),(t=5.7, p<0.05). But the iNOS expression in these pyknotic cells elevated.ConclusionsThe EGCG inhibited the proliferation of A549cells and stimulating the apoptosis may be mediated by reduced the expression of iNOS protein and mRNA.