Glycogen Synthase Kinase-3β (GSK-3β) Involved In The Toxic Effect Induced By Trichostatin A On Lung Carcinoma Cells In Vitro

ObjectivesTo explore the effect and the potential mechanisms of glycogen synthase kinase-3β(GSK-3β) on trichostatin A (TSA)-induced growth inhibition, cell cycle arrest and tumor cells invasion and metastasis inhibition in human lung adenocarcinoma cell line A549.MethodsA549 cells were regularly subcultured in vitro and were divide into groups with treatments by different concentrations of trichostatin A(TSA) and pre-treatment with SB216763 (a specific inhibitor of GSK-3β) , the cells were seeded for 24h. MTT assay was employed to evaluate the effects of different concentrations of TSA on the proliferation of A549 cells and observe the above-mentioned concentrations of TSA on the growth of A549 cells with SB216763 inhibited GSK-3βactivity. The use of enzyme inhibitors before and after intervention in A549 cells, TSA on cell cycle of A549 was examined by flow cytometry analysis. The transwell chambers were employed to evaluate the invasion of A549 cells in groups that GSK-3βinhibitory activity before and after. Western Blot was employed to analyze the protein levels of GSK-3β, phosphorylated GSK-3β(pGSK-3β) and adhesion molecule E-cadherin in different concentrations of TSA on the A549 cells and that pre-treatment or not with SB216763.The immunocytochemical method was employed to analyze the protein levels of cyclin-dependend kinase inhibitor p27 in cells in each group that the use of enzyme inhibitors before and after.Results1,MTT assay showed TSA could inhibit the proliferation of A549 cells in a dose-dependent manner and the cell cycle was arrested in G0/G1 phase in A549 cells treated with TSA(P<0.05);While pre-treatment of A549 cells with SB216763 attenuated TSA induced growth inhibiting and cell cycle arrest(P<0.05).2,The transwell chambers showed that A549 cells had a certain degree of invasiveness in vitro, after treatment with TSA the cell invasions were lower than that in control group(P<0.05). With the increase in TSA concentration, A549 cells reduced the invasiveness of the more obvious. After pretreatment with SB216763,the invasion of A549 cells increased than TSA alone (P<0.05).3,A549 cells had a abundant expression of GSK-3β,TSA did not alter the levels of GSK-3βprotein, there was no change in the expression levels of pGSK-3βand p27 protein in A549 cells treated with 12.5ug/L TSA(P>0.05).TSA decreased pGSK-3βexpression and up-regulated p27 and E-cadherin protein level form 25ug/L, (P<0.05). As expected, SB216763 increased the levels of pGSK-3β, consistent with inhibition of GSK-3βactivity. Inhibition of GSK-3βactivity down regulated E-cadherin and p27 protein level (P<0.05).Conclusion1,GSK-3βwas involved in the growth inhibition and cell cycle arrest effect induced by TSA on lung carcinoma cells. The mechanism may be related to GSK-3βon the regulation of cyclin-dependend kinase inhibitor p27 protein.2,TSA degraded lung carcinoma cells invasive power by up-regulated GSK-3βactivity, GSK-3βincreased tumor metastasis suppressor molecule E-cadherin protein level ,which may be the mechanism of TSA inhibiting the metastasis of Human Lung Adenocarcinoma Cell line .3,Inhibitory activity of GSK-3βdrugs coudle reduce the anti-cancer role of the chemotherapy on lung adenocarcinoma,GSK-3βmay be a new target for chemotherapy .