Experimental Study To The Effects Of SCD40L Combined With LY29400 On The Proliferation And Invasion Of AGS Gastric Cancer Cells

PartⅠEffect of sCD40L on the proliferation of AGS gastric cancer cellsObjective To investigate the effects of soluble CD40 ligand, on the growth and apoptosis of AGS gastric cancer cell.Methods The growth of AGS cells treated by different concentrations of sCD40L was measured by MTT assay. Changes of cells cycle and apoptosis rate of AGS cells were analyzed by flow cytometry (FCM).Results After being cultured with sCD40L, the growth of AGS cells was significantly inhibited in dose and time dependent manner (P<0.05). Flow cytometry displayed that AGS cells G1 phase was arrested in dose dependence after they were incubated 48 hours with sCD40L (P<0.05), but AGS cells had no obviously apoptosis.Conclusion sCD40L can significantly inhibit the growth of AGS cells in a dose-and-time dependent manner by arresting cells cycle.PartⅡEffect of LY294002 on the proliferation of AGS gastric cancer cellsObjective To investigate the effects of LY294002, a specific inhibitor of phosphatidy- linositol 3-kinase, on the growth and apoptosis of AGS gastric cancer cells.Methods The growth of AGS cells treated by different concentrations of LY294002 was measured by MTT assay. Changes of cells cycle and apoptosis rate of AGS cells were analyzed by flow cytometry (FCM).Results After being cultured with LY294002, the growth of AGS cells was significantly inhibited in dose and time dependent manner (P<0.05). Flow cytometry displayed that LY294002 can raise AGS cells apoptosis and induce G1 phase arrest in dose dependence after they were incubated 48 hours with LY294002 (P<0.05).Conclusion LY294002 can significantly inhibit the growth of AGS cells in a dose- and- time dependent manner by arresting cells cycle and inducing cells apoptosis.PartⅢEffects of sCD40L combined with LY294002 on the proliferation and invasion of AGS gastric cancer cellsObjective To investigate the effects and mechanisms of sCD40L (soluble CD40 ligand) combined with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase, on proliferation and invasion of AGS gastric cancer cells.Methods The MTT assay was used to analyze the effect of sCD40L(2ug/ml)combined with LY294002(20μmol/L)on cells growth, the Flow cytometry (FCM) was used to analyze cells cycle and apoptosis, Election microscope was used to observe the cells morphologic change, Would healing and Transwell invasion assay were used to evaluate the abilities of cells invasion and migration, Western blot analysis was used to evaluate the cells protein expression of PI3K,p-Akt and VEGF.Results (1)MTT assay showed that both sCD40L and LY294002 could effectivly inhibit AGS gastric cancer cells growth, sCD40L combination with LY294002 had synergistic effect (P<0.05).(2)Flow cytometry displayed that both sCD40L and LY294002 could effectivly induce AGS cells G1 phase arrest, the effect was enhanced after being treated by LY294002 combined with sCD40L(P<0.05). LY294002 can raise cells apoptosis, while the cells treated by sCD40L had no obviously apoptosis, comparing with LY294002 alone, the rate of cells apoptosis increased after being treated by LY294002 combined with sCD40L(P<0.05).(3)Election microscope showed that after being treated by LY294002 combined with sCD40L, cellular nucleus became condensed, heterochromatin agglutinating, showing typical apoptosis changes in morphology, while the cells treated by sCD40L had no obviously apoptosis changes in morphology.(4)Would healing and Transwell invasion assay showed that comparing with LY294002 and sCD40L alone, the inhibitory effect of cells migration and invasion was increased after they were treated by LY294002 combined with sCD40L (P<0.05)(.5)Western blot analysis indicated that AGS cells protein expression of PI3K,p-Akt was upregulated after being treated with sCD40L, Contrary, the cells protein expression of PI3K,p-Akt was downregulated after being treated by LY294002 and LY294002 combined with sCD40L, the cells protein expression of VEGF was downregulated when cells were treated by LY294002 or sCD40L, while the cells protein expression of VEGF was downregulated more when cells were treated by LY294002 combined with sCD40L(P<0.05).Conclusion CD40 stimulation antagonized the anti-gastric cancer effect by activing PI3K/Akt signaling pathway, blocking PI3K/Akt signaling pathway can significantly enhance the sCD40L inhibitory effects on growth and invasion of AGS gastric cancer cells.