The Effects And Mechanisms Of LY294002 Enhances Gastric Cancer 5-fluorouracil Chemosensitivity

AimTo examine the effects of combined treatment of 5-fluorouracil (5-FU) and phosphatidylinositol 3'-kinase (PI3K) inhibitor,2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) for gastric cancer.Methods(1) BGC823 and MGC803 human gastric cancer cell lines were treated with various doses of 5-FU (0,5,10,20μM) for 24 h with or without the pretreatment of LY294002 (20μM). Apoptotic cells were detected by flow cytometric analysis. Invasion of treated cells was assessed using Matrigel. Migration of treated cells was assessed using wound healing assays.(2) BGC823 and MGC803 human gastric cancer cell lines were treated with various doses of LY294002 (0,1,10,20μM) for 24 h with or without the treatment of 5-FU (20μM). Apoptotic cells were detected by flow cytometric analysis. Invasion and migration of treated cells were assessed using Matrigel and wound healing assays.(3) Western blotting was used to examine protein expression of phosphorylated protein kinase B (AKT)-Ser473(p-AKT-Ser473),p-FKHR-Ser24,p-FKHRL1-Thr253,Fas ligand (FasL),caspase-8/FLICE-inhibitory protein (c-FLIP) matrix-metalloproteinases 2 (MMP-2),MMP-9,tissue inhibitor of matrix metalloproteinase-1 (TIMP-1),TIMP-2,membrane-type matrix-metalloproteinase 1 (MT1-MMP),MT2-MMP, the activation of caspase-8,caspase-3, the cleavage of Bid (t-Bid). Immuno-precipitation was used to examine Fas-associated death domain protein (FADD) recruitment. Nuclear factor kappa B (NFκB) binding activities were investigated using electrophoretic mobility shift assay (EMSA). The activation status of MMP-2 and MMP-9 were investigated by gelatin zymography. (4) BGC823 and MGC803 cells transfected with FasL siRNA were treated with 5-FU, LY294002, or a combination of both. Apoptotic cells were detected by flow cytometric analysis.(5) To further investigate the effect of 5-FU, LY294002, or a combination of both treatment on the tumor growth and apoptosis of gastric cancer cells in vivo, BGC823 cells were subcutaneously implanted into the nude mice. Hnmunohistochemistry were used to examine the expression of p-AKT-Ser473-. p-FKHR-Ser24,p-FKHRL1-Thr253, and the expression of the death receptor pathway molecules.(6) To further investigate the effect of 5-FU, LY294002, or a combination of both treatments on the tumor metastasis of gastric cancer cells in vivo, the nude mouse orthotopic transplantation model was used. Hnmunohistochemistry were used to examine the expression of NFκB-p65,MMP-2,MMP-9,TIMP-1,TIMP-2 MT1-MMP,MT2-MMP.Results(1) Treatment with 5-FU resulted in enhancement of cell apoptosis, inhibition of invasion and migration in vitro, while LY294002 increased this effect of 5-FU (P< 0.05).(2) Treatment with LY294002 resulted in enhancement of cell apoptosis, inhibition of invasion and migration in vitro, while 5-FU increased this effect of LY294002 (P< 0.05).(3) In BGC823 and MGC803 cells,5-FU treatment inhibited AKT,NFκB,FKHR,FKHRL1 activation, while pretreatment with LY294002 enhanced 5-FU-induced inhibition of AKT,NFκB,FKHR,FKHRL1.(4) LY294002 promoted 5-FU-induced FasL expression, FADD recruitment, caspase-8,Bid and caspase-3 activation, and the short form of c-FLIP (c-FLIPs) inhibition. In addition, the active forms of MMP-2 and MMP-9 were greatly reduced in BGC823 and MGC8O3 cells treated with combination of LY294002 and 5-FU than those treated with 5-FU alone. Compared with that of 5-FU treatment alone in BGC823 and MGC893 cells, the combination of 5-FU and LY294002 significant decreased the levels of TIMP-1,TIMP-2 and increased the levels of MT1-MMP.(5) FasL expression was inhibited by FasL siRNA in BGC823and MGC803 cells. FasL silencing decreased LY294002,5-FU-or combination-induced cell apoptosis.(6) When compared with 5-FU treatment alone,5-FU combined with LY294002 significantly inhibited tumor growth and enhanced cell death in the tumor mass via apoptosis (P< 0.05). Immunohistochemical analysis was performed to evaluate the expression of death receptor pathway molecules. LY294002 increased 5-FU-induced inhibition of p-AKT-Ser473,p-FKHR-Ser24,p-FKHRL1-Thr253, and increased 5-FU-induced expression of FasL, inhibition of c-FILPs, and activation of casepase-8,Bid, and caspase-3.(7) The numbers of hepatic or lung metastatic lesions in the LY294002+5-FU group were much lower compared with 5-FU group (P< 0.05). Moreover, the metastatic lesions in the LY294002+5-FU groups were much smaller than that in the 5-FU group. The protein expression of NFKB-p65,MMP-2,MMP,TIMP-1,TIMP-2,MT1-MMP,MT2-MMP in nude mouse xenograft were detected by immunohistochemical analysis. LY294002 increased 5-FU-induced inhibition of NFKB-p65,MMP-2,MMP-9,TIMP-1,TIMP-2,and increased 5-FU-induced expression of MT1-MMP.Conclusions(1) LY294002 increased 5-FU-induced enhancement of apoptosis, and inhibition of invasion and migration in vitro.(2) LY294002 increased 5-FU-induced inhibition of tumor growth and metastasis, and enhancement of apoptosis in the tumor mass in vivo. (3) The death receptor pathway might be involved in the cell apoptosis induced by 5-FU or LY294002 in gastric cancer cells.(4) Through the expression and activation of MMP-2 and MMP-9, LY294002 increased 5-FU-induced inhibition of metastasis.(5) Combination of 5-FU and LY294002 was therapeutically promising for gastric cancer treatment.