Positive expression of Akt, p Akt, and caspase 9 locates in the cytoplasm. Immunohistochemical expression of Ki67 and TUNEL positive cells shows in the nuclei. The mean per centage of positive tumor cells was determined from five areas at highpower field. The growth index and the apoptosis index were calculated by counting the Ki67 and TUNEL positive cells in a total of 1000 tumor cells observed from more than representative highpower fields, respectively. Immunohistochemical results were evaluated independently. Statistical analysis Data were expressed as mean SD of mean and com pared by unpaired Students t test. ELISA Assay was used by the Linear Regression. Results were considered signifi cant at a value of P 0. 05.

Results Effects of PI3K/Akt inhibition on proliferation and apoptosis of NPC cells To determine whether inhibition of PI3K/Akt activ ity would inhibit cell proliferation and pro mote apoptosis in CNE 2Z cell line, MTT assay and flow cytometry analysis were used. When the cells were cul tured in medium containing different concentrations of LY294002 for 24 h and 48 h, cell proliferation was remarkably decreased in a dose dependent fashion. The Annexin V/PI assay was used to detect apoptosis in NPC cells. As shown in Fig 2A, the proportion of apop totic cells was significantly increased in dose dependent. Caspase 9 is regarded as a most likely candidate relating with apoptosis induced by PI3K inhibitor, we explored whether caspase 9 was involved in LY294002 induced cell apoptosis in CNE 2Z cells by detecting cas pase 9 activity in cells treated with PI3K/Akt inhibitor.

When caspase 9 specific inhibitor, ZVAD, was added, apoptosis rate was decreased Carfilzomib 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 Akt were decreased in treated NPC cells, exhibiting a dose response effect. 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 expression levels were significantly decreased in treated group. At the same time, 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. Effects of PI3K/Akt inhibition proliferation and apoptosis in vivo Tumors generated by orthotopic implantation of the met astatic CNE 2Z cell line were used to evaluate the effect of LY294002 on proliferation and apoptosis in an orthoto pic xenograft model. All of the mice were sacrificed after 4 weeks of treatment.