se using the Mascot program. The following parameters were used for database searches, taxonomy, Homo sapiens, cleavage specificity, trypsin with one missed cleavage allowed, peptide tolerance of 100 ppm for the fragment ions, and allowed modifica tions, Cys Carbamidomethyl, and oxidation of Met. Protein scores 56 were considered statistically significant. Western blot analysis AGS cells were cultured in 6 well plates and incubated with vitamin C at 300 ug mL or PBS as the solvent control for 24 h. After incubation, cells were washed with ice cold PBS and lysed with a lysis buffer, containing the protease inhibitor cocktail. The cell debris was removed by centrifugation at 13,000 rpm for 30 min and protein con centration was determined using a Bradford assay.

Proteins were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to a polyvinyldene fluoride membrane using the TE Cilengitide 77 Semi Dry Transfer Unit. The membrane was blocked with 5% non fat skim milk in Tris buffered saline containing 1% Tween 20 at room temperature for 1 h, and the blots were probed with rabbit monoclonal antibody to 14 3 3��, 14 3 3�� and 14 3 3, and mouse monoclonal antibody for B actin. The proteins were visualized using an enhanced chemiluminescence kit and Western blotting detec tion reagents, and exposed to X ray film. Each band was quanti tatively determined using Image J software. The densitometry readings of the bands were normalized to B actin expression. Statistical analysis The data represents the mean standard deviation of three independent experiments.

The statistical signifi cance between the control and sample groups was cal culated by the Students t test. A p value 0. 05 was considered as significant. Results Growth inhibition of AGS cells by vitamin C To evaluate the effects of growth inhibition and survival of AGS cells, the AGS cells were cultured in the pres ence of various concentrations of vita min C for 24 h. Vitamin C had a strong inhibitory effect on cell proliferation of AGS cells in a dose dependent manner when compared to the control, after 24 h treat ment with vitamin C. Especially, vitamin C at 300, 400 and 500 ug mL decreased the cell growth by approximately 50%, 36% and 27%, respectively. Therefore, the IC50 of vitamin C was found to be approximately 300 ug mL.

Moreover, microscopic observations revealed morphological changes in AGS cells, such as cell shrinkage and dens ity compared with the control cells. Further, 2 DE gel analysis was performed to study the protein expressions in AGS cells due to inhibitory effects of vitamin C. Proteomic analysis to identify differentially expressed proteins in vitamin C treated AGS cells We performed a proteomic approach to identify proteins that were differentially expressed in vitamin C treated AGS cells, 100 ug of total proteins were sepa rated by IEF on 18 cm IPG strips in the first dimension and 12% SDS PAGE in the second dimension. We ob served a total of approximately 500 protein spot