All samples were assayed in triplicate, and the mean for each experiment was cal culated. Results were expressed as a percentage of con trol, which was considered to be 100%. Annexin V/PI for cell apoptotic analysis Cells were collected with 0. 25% trysin/0. 02% EDTA after presence of LY294002 24 h and 48 h. At the same time, caspase 9 specific inhibitor, ZVAD, was added for 48 h. Cells were harvested at the end of treatment, rinsed twice with PBS, and stained with Annexin V FITC apoptosis detec tion kit I. Analysis was performed on the FACS Calibur using CellQuest software. P Akt ELISA assay CNE 2Z cells were plated on 6 well plates in RPMI 1640 plus 10% FBS in duplicate for each treatment. Chemical inhibitor LY294002 was added to the appropriate wells. The cells were incubated at 37 C for 24 h and 48 h.

Phos phorylated protein level of treated and untreated cells lysates was measured using a commercially available ELISA kit. Statistical analysis to determine significance of the observed differences was used by the Linear Regres sion. Experiments were repeated three times. Western blot analysis Cells were homogenized in 500 ul with lysis buffer, 0. 1% SDS, 150 mM NaCl, 10% glycerol, 1. 5 mM MgCl2, 1 mM PMSF, 0. 1 mM Na V04, 0. 1 mM benzamidine, 5 ul/ml leupeptin, 5 ul/ml aprotinin. The lysates were clarified by centrifuga tion at 12000 g for 15 min at 4 C. Samples were analyzed by 15% SDS polyacrylamide gels, and transferred to nitrocellulose membranes, and the membranes was incu bated with primary antibodies, followed by horseradish peroxidase cunjugated secondary antibodies.

An anti body for B actin was used as a loading control. Tumor xenograft experiments All of the experiments involving animals in present study were approved by the animal center of Guangdong Medi cal College in accordance to institutional and Guangdong government guidelines for animal experiments. Athymic nude mice were used when they were 6 8 weeks. Mice were randomly divided into free separated into five groups. Mice were housed in the same envi ronment with controlled temperature, humidity, and a 12 h light/dark cycle. Mice were inoculated subcutaneously with CNE 2Z cells into the flank. The tumor take rate was 100%. After 1 week, an intraperitoneal Batimastat injection was per formed to the xenograft mice with different dosage of LY294002, 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 volume was calcu lated. 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 were used for immunohistochemical analysis of Akt, p Akt, caspase 9, Ki67, and the TUNEL method for determining of DNA fragmentation.