Athymic nude mice were used when they were 6-8 weeks. Mice were randomly divided into free separated into five groups (n = 4 mice). Mice were housed in the same environment with controlled temperature,
humidity, and a 12 h light/dark cycle. Mice were inoculated subcutaneously with CNE-2Z cells (1 × 106 cells/mouse in 200 μl of RPMI-1640) into the flank. The tumor take rate was 100%. After 1 week, an intraperitoneal injection was performed to the xenograft mice with different dosage of LY294002 (10 mg/kg, 25 mg/kg, 50 mg/kg, and 75 mg/kg twice weekly (n = 4 mice), each group for 4 weeks. Treated mice were monitored any signs. Body weight and tumors size were measured twice a week. Tumor size was measured using calipers and tumor SGC-CBP30 ic50 volume was calculated (volume = long axis × short axis2). At the end of the treatment, all mice were euthanized. One part of tumor tissue was fixed in formalin and embedded in paraffin, and another part was stored at -70°C. Immunohistochemistry analysis Paraffin sections selleck kinase inhibitor were used for immunohistochemical
analysis of Akt, p-Akt, caspase-9, Ki67, and the TUNEL method for determining of DNA fragmentation. TUNEL assay was carried out according to the protocol of the ApopTag Peroxidase in situ apoptosis detection kit (Chemicon International, selleck chemical Temecula, Calif, Methane monooxygenase USA). 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 percentage of positive tumor cells was determined from five areas at highpower field (×400). The growth index (GI) and the apoptosis index (AI) 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 compared by unpaired Student’s t test. ELISA Assay was used by the Linear Regression. Results were considered significant 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 activity(LY294002) would inhibit cell proliferation and promote apoptosis in CNE-2Z cell line, MTT assay and flow cytometry analysis were used. When the cells were cultured in medium containing different concentrations of LY294002 for 24 h and 48 h, cell proliferation was remarkably decreased in a dose-dependent fashion (Fig 1). The Annexin V/PI assay was used to detect apoptosis in NPC cells. As shown in Fig 2A, the proportion of apoptotic cells was significantly increased in dose-dependent.