When caspase-9 specific inhibitor, ZVAD, was added, apoptosis rate was decreased at 48 h (Fig 2B). Figure 1 CNE-2Z cells growth rate in different concentrations of LY294002. Figure 2 CNE-2Z cells apoptosis rate. CNE-2Z cells apoptosis rate induced by different concentrations of LY294002. B. CNE-2Z cells apoptosis inhibited by different concentrations of ZVAD (0, 5, 10, and 20 μmol/L) at 48 h. Effects of PI3K/Akt inhibition on Akt phosphorylation in NPC Cells When LY294002 was added to NPC cells with different concentrations, levels of phosphorylation (S473) Akt were decreased in treated NPC cells, exhibiting a dose-response effect (Table 1).

Table 1 Expression of p-Akt CH5183284 order protein in CNE-2Z cells treated with LY294002 LY294002 (mol/L) n P-Akt(unit/ml) 0 3 74.10 ± 1.00 10 3 62.65 ± 0.68 25 3 50.09 ± 1.83 50 3 25.22 ± 1.83 75 3 13.21 ± 1.34

F   1328.43 P   < 0.001 Effects of PI3K/Akt inhibitionon protein expression in NPC cells The results of Western blot showed that total Akt protein level was not difference with different concentration. In contrast, phosphorylated Akt (S473) expression levels were significantly decreased in treated group. At the same time, Proteasome structure we explored whether caspase-9 was involved in LY294002- induced cell apoptosis in CNE-2Z cells by detecting caspase-9 activity in cells treated with PI3K/Akt inhibitor. The results show caspase-9 activity in CNE-2Z cells was up-regulated by LY294002, whereas the level of caspase-9 was not changed after using ZVAD (Fig 3). Figure 3 Western blot analysis of Akt, phosphor-Akt(S473), caspase-9, and caspase-9 treated with ZVAD (20 μmol/L). N: no treatment group; Lanes1, 2, 3, and 4: treatment with LY294002(10 μmol/L, 25 μmol/L, 50 μmol/L, and 75 μmol/L respectively). Effects of PI3K/Akt

inhibition ITF2357 proliferation and apoptosis in vivo Tumors generated by orthotopic implantation of the metastatic CNE-2Z cell line were used to evaluate the effect of LY294002 on proliferation much and apoptosis in an orthotopic xenograft model. All of the mice were sacrificed after 4 weeks of treatment. Treatment with LY294002 (50 mg/kg, 75 mg/kg) significantly reduced mean NPC tumor burden as compared with the control group (LY294002 50 mg/kg, 75 mg/kg; P < 0.001). Treatment with 10 mg/kg or 25 mg/kg LY294002 was less effective in decreasing tumor burden. Mean NPC tumor burden treated with LY294002 was remarkably decreased in a dose-dependent manner, whereas mean body weight was no obvious difference between control and treated groups (LY294002 10 mg/kg, 25 mg/kg, 50 mg/kg, and 75 mg/kg; P > 0.05; Fig 4A and 4B). Compared with control, TUNEL-positive cells treated with LY294002 were significantly increased in a dose-dependent fashion (Fig 4C and 4D), with significant difference (P < 0.01). Figure 4 Growth and apoptosis analysis of tumors xenografts in athymic nude mice. A. Mean body weight and NPC tumor burden treated with LY294002. B.