find the biomarkers of gastric cancer. However, no 2 DE proteome of vitamin C treated AGS cells have hitherto been reported. Our previous study demonstrated that vitamin C in duced apoptosis in human adenocarcinoma AGS cells at pharmacological concentrations, and inhibited AGS cells proliferation. In the present study, we perform a proteome analysis of AGS cells treated with vitamin C at pharmacological concentrations and the control, and 20 different expressed proteins were identified by MALDI TOF MS. Also, the expression of isoforms of 14 3 3 proteins was confirmed by immuno blotting. The cytotoxicity assay suggests that vitamin C inhibited AGS cells growth and proteome results re vealed that apoptosis related proteins were involved in promoting and regulating cell death of AGS cells.
Methods Chemical and reagents RPMI 1640 medium was purchased from Hyclone. Fetal bovine serum Cilengitide and antibiotics were purchased from Gibco. Materials and chemicals used for electrophoresis were obtained from BioRad. Antibody to 14 3 3�� and B actin were purchased from Millipore. 14 3 3�� and 14 3 3 were obtained from Bioworld Tech nology Inc. Vitamin C was provided by Animal Resources Research Bank. All other chemicals used in this study were purchased from AMRESCO and Sigma Aldrich. All the chemicals used were of the highest grade commercially available. Cell culture and treatments AGS human gastric cancer cell line was purchased from ATCC. Cells were grown in RPMI 1640 medium supplemented with 10% FBS and 1% peni cillin streptomycin, and grown in a humidified in cubator with 5% CO2 in air at 37 C.
Experiments were performed when cell growth was approximately 80% confluent. Cytotoxicity assay The 3 2, 5 diphenyltetrazolium bromide based assay was performed to determine the cytotoxicity of vitamin C on AGS cells. Cells were seeded at 10 �� 104 cells mL in a 12 well plate and incu bated for 24 h. Cells were treated with various concentra tions of vitamin C or only vehicle and incubated for 24 h. After incubation, 100 ul of a MTT solution was added to the wells and incubated for 3 h. Then, 500 ul of di methyl sulfoxide was added to each well after the medium was removed completely to dissolve the cellular crystalline deposits. The optical density was measured at 540 nm using an ELISA plate reader.
Protein extraction and two dimensional gel electrophoresis A total of 1��107 cells was plated onto 100mL plates and incubated overnight at 37 C in an atmosphere of 5% CO2. Cells were treated with 300 ug mL of vitamin C and 1X PBS used as the control. After 24 h incubation, cells were trypsinized and washed twice with cold 1X PBS. Then, cells were lysed in a lysis buffer CHAPS on ice for 1 h. The lysates were centrifuged at 14000 rpm for 15 min at 4 C, and the col lected supernatant was stored at ?80 C until analysis. Pro teins in lysates were precipitated with equal volume of 20% v v trichloroacetic acid and dissolved in 7 M urea, 2 M thiourea, and 4% CHAPS, 0. 5% IPG buffer, and