Effects Of Glycogen Synthase Kinase 3β On Cell Proliferation And Apoptosis Of Human Lung Adenocarcinoma Cell Line A549

Background and Objective Lung cancer is the leading cause of death from cancer worldwide, with its incidence continuing to rise. Non-small cell lung cancer (NSCLC) is a major type and accounts for about 80% of all lung cancer. Despite advances in chemotherapy, multimodality strategies, using conventional therapies only minimally improve survival rates even in early stages of lung cancer. Thus, newer agents must be developed to establish an effective therapeutic strategy against NSCLC.The disbalance between proliferation and apoptosis signals contributes to the pathogenesis of a variety of cancers. Glycogen synthase kinase 3 (GSK3), a serine/threonine phosphokinase which has extensive substrates, plays important roles in a variety of life activities such as cell proliferation, differentiation and apoptosis. There are two mammalian GSK3 isoforms, designated GSK3αand GSK3β. GSK3 is constitutively active in resting cell, and can be inactivated by phosphorylation of Ser-21 in GSK3αor Ser-9 in GSK3β. A recent investigation indicated that the development of lung cancer was closely related to dysregulation of Wnt signals. Though GSK3 is a key factor in Wnt signals and meanwhile abundantly expressed in alveolar epithelium, little is known about the role of GSK3 in lung carcinogenesis. Therefore, in the present study, we used human lung adenocarcinoma cell line A549 to investigate the effects of GSK3βon cell proliferation and apoptosis.Method After treatment of LiCl, a selective inhibitor of GSK3β, or transient transfection of GSK3βmutant plasmids, CCK-8 assay/cell count were made to detect the effect of GSK3βactivity changes on cell proliferation of A549; cell cycle analysis was proceeded by flow cytometry; morphologic observation was made by immunofluorescence technique and confocal microscopy; AnnexinⅤ/PI dual staining, as well as FITC-labeling TUNEL assay were used for detection of apoptosis; the expression changes of associated proteins were determined by western blot; Co-immunoprecipitation was made to examine whether there was a direct interaction between GSK3βand survivin.Result Firstly, A549 cells were treated with lithium chloride (LiCl), and then, the effects of LiCl on A549 cell growth were observed. Results: (1) CCK-8 assay showed that LiCl promoted cell proliferation of A549 (P<0.05) by a dose-dependent manner. That is, the cellular proliferation rate is accelerated accompanied by the increase of LiCl concentration within a certain extent (1~20 mmol/L). (2) Flow cytometry demonstrated that 10 mmol/L LiCl treatment caused a obvious decrease of cell population in G1 phase (P<0.05) while a notable increase of the cell amount in S phase (P<0.05); Consistent with that, 20 mmol/L LiCl treatment resulted in a similar and more significant changes about distribution of each cell cycle phase ((P<0.01, respectively). (3) Immunofluorescence showed weakened fluorescence of GSK3βin A549 cells after 10 mmol/L or 20 mmol/L LiCl treatment for 24 h, suggesting GSK3βis possibly involved in growth-inhibitory effect of LiCl.In order to further determine that GSK3βcontributes to A549 cell growth, we directly changed the activity level of GSK3βby transient transfection of GSK3βmutant plasmids. And then, morphologic observation, cell counting, as well as cell cycle analysis were made subsequently. Results: (1) Under contrast phase microscope, the empty vector-transfected control cells looked spindle to round and stereo with close intercellular adhesion . Compared with them, the DN-GSK3β-transfected cells did not manifest distinct morphological changes except an increase in cell number. Contrary to that, there was a great decrease in cell number in S9A-GSK3βgroup, and the cells became flattened, elongated and shrunken. Additionally, a further cell counting signified S9A-GSK3βtransfection might inhibit A549 cell proliferation while DN-GSK3βtransfection promote cell growth (P<0.05, compared with control group, respectively). (2) compared with the control group, the percentage of cells in G1 phase was significantly increased (P<0.01), whereas the population of cells in the S phase was decreased (P<0.05) in constitutively active S9A-GSK3β-transfected group; however, in dominant negative DN-GSK3β- transfected group, there was a decreased population of cells in G1 phase (P<0.05), with an accompanied increased population of cells in S phase (P<0.05).Meanwhile, by western blot, we found that both LiCl treatment and transient transfection of DN-GSK3βmutant plasmids were able to up regulate Cyclin D1 expression, while transient transfection of constitutively active S9A-GSK3βdominantly down regulate expression of Cyclin D1.Furthermore, we also studied the effects of GSK3βon cell apoptosis of A549. After LiCl treatment or transient transfection of GSK3βmutants for 24 h, A549 cells were processed by AnnexinⅤ/PI dual staining and analysis by flow cytometry. The results showed that compared with corresponding control, 10 mmol/L,20 mmol/L LiCl as well as dominant negative DN- GSK3βtransfection all certainly inhibited the apoptosis of A549 cells, while transient transfection of constitutively active S9A-GSK3βapparently elevated apoptotic rate(P<0.05). By FITC-labeling Fluorometric TUNEL System and confocal microscopy, we found that S9A-GSK3β-transfected group obviously showed a higher apoptotic rate than that of WT-GSK3βgroup (P<0.05). The apoptotic cells demonstrated green fluorescence, with nuclei condensed or fragmented, and sometimes formation of apoptotic bodies. The above data suggested that GSK3βplayed a proapoptotic role in A549 cells.To investigate whether survivin is involved in the proapoptotic effect of GSK3β, we detected survivin protein expression in A549 cells after LiCl treatment or transient transfection of GSK3βmutants. Western blot showed that the expression level of survivin protein is elevated by LiCl treatment in a dose-dependent manner. After transient transfection of GSK3βmutant plasmids for 24 h, compared with the control group, DN- GSK3βpresented a similar effect of LiCl treatment, i.e., up-regulation of survivin expression in A549 cells; conversely, constitutively active GSK3βmutant greatly decreased survivin protein expression.More intriguingly, by multiple immunofluorescence technic and confocal microscopy, we surprisingly found an important phenomenon: with comparison to control group, besides decreased protein expression of survivin, there was also obvious nuclear translocation of survivin in A549 cells in S9A-GSK3βtransfected group. Moreover, LY294002, an inhibitor of protein kinase B (PKB, an upstream kinase of GSK3β), which can promote GSK3βactivity, demonstrated a similar effect. The above both implicate that elevating GSK3βactivity may induce nuclear translocation of survivin.A further co-immunoprecipitation test showed that whether by anti-GSK3βantibody or by anti-survivin antibody, we always could find the presence of the other protein (survivin or GSK3β) in the precipitate, suggesting there might be a direct action between the two proteins.Conclusion (1) GSK3βcan inhibit cell proliferation in A549 cells. (2) GSK3βmay exert its proliferation-inhibitory effect by down regulating Cyclin D1 and thus inducing G1 cell cycle arrest. (3) GSK3βplays a proapoptotic role in A549 cells. (4) Down regulation of survivin expression as well as nuclear translocation may contribute to the proapoptotic effect of GSK3βin A549 cells.This study promote a possibility that GSK3βmight be a critical downstream effector of the antitumor effects. Therefore, potential approaches for the development of pharmacological agents designed to increase GSK3βactivity may lead to new treatment strategies in lung cancer therapy.