s in silver stained gels. Control and vitamin C treated gels were analyzed by using Progenesis Samespots software, and we found 32 statistically significant differentially expressed protein spots. These 32 differentially expressed pro teins spots were chosen for further analysis by MALDI TOF MS. Finally, 20 differentially expressed proteins were successfully identified by using the MASCOT search en gine and the SwissProt database. Of these 20 proteins, six were up regulated and fourteen were down regulated in vitamin C treated AGS cells com pared with the control. Cilengitide Down regulated proteins involved in cell motility included tropomyosin alpha 3 chain and tropomyosin alpha 4 chain, whereas Xin actin binding repeat containing protein 1 was up regulated.
In addition, the peroxiredoxin 4 and thioredoxin domain containing protein 5 were involved in antioxidant and detoxification, which was up regulated. While proteins participating in signal transduction were significantly down regulated, includ ing 14 3 3 protein sigma, 14 3 3 protein epsilon and 14 3 3 protein zeta delta, whereas TNFAIP3 interacting protein 2 was up regulated. Proteins involved in protein metabolism Eukar yotic translation initiation factor 3 subunit K, Proteasome subunit alpha type 5 and Prote asome subunit beta type 6 were down regulated. Further, we showed the enlarged 2 DE images of 6 import ant protein spots, one spot was up regulated and the other five were down regulated in the vitamin C treated AGS cells compared with the control.
Validation of expression of 14 3 3 isoforms by immunoblotting Recent research on cancer targets have focused 14 3 3 pro teins that are known to be involved in various biological processes like signal transduction, cell cycle control, apop tosis, cellular metabolism, proliferation, cytoskeletal regula tion, transcription, and redox regulation or stress response. The AGS cells were treated with vitamin C and the expression of 14 3 3��, 14 3 3�� and 14 3 3 were examined by immuno blotting. Quantification of the protein bands revealed that the expression of 14 3 3��, 14 3 3�� and 14 3 3 were decreased in the vitamin C treated group compared to the vehicle treated control group. These data indicated that vitamin C decreased the expression of 14 3 3 isoforms in AGS cells. Discussion Apart from antioxidant activity, vitamin C plays an effect ive role of cancer prevention and treatment.
The numerous studies have reported that vitamin C prevents cell prolifer ation and metastasis of many human cancer cells. But, its exact molecular mechanisms still has not been fully elucidated. In the previous study, we demonstrated that vitamin C at pharmacological concentration induced apop tosis in AGS cells, mainly through the down regulation of 14 3 3�� protein and dephosphorylation Bad proteins via a mitochondrial dependent pathway. In the present study, we performed 2 DE analysis coupled with MALDI TOF MS of AGS cells treated with vitamin C at a pharma cological concentrati