The Screening And Analysis Of The Protein Signatures Associated With Signal Pathway In Gastic Cancer And The Effect Of XIAP And CDK6 Inhibitors On Gastic Cancer Cells

Objection:Identification of the protein signatures associated with signal pathway in gastic cancer through Protein Pathway Array, and study the effect of XIAP and CDK6 inhibitors on gastic cancer cells.Methods:In the first part, Protein Pathway Array was used to assess the protein expression of 149 fresh frozen gastric samples (56 paired tumor and normal tissues and 37 unpaired tumors). Fifty six paired samples were divided into a training set (n=37) and a validation set (n=19) for the identification differentially expressed proteins between tumor and normal tissues. Fifty six tumor samples were used to identify proteins correlating with T/N staging. All of the ninety three tumor samples were used to identify candidate proteins for predicting survival. In the second part, we determine the effect of Embelin on gastric cancer cell viability through MTT assay. To determine whether the growth inhibition of gastric cancer cells by Embelin was associated with cell cycle arrest or apoptosis, flow cytometry were performed. We further examined the expression of cellular proteins of the cell cycle regulating components through Westenblot,.we performed protomic-wide expression analysis using Protein Pathway Array and Ingenuity Pathway Analysis(IPA)software to investigate differences in protein expression between Embelin treated and untreated gastric cancer cells.In the third part, we determine the effect of PD0332991 on gastric cancer cell viability through MTT assay. To determine whether the growth inhibition of gastric cancer cells by PD0332991 was associated with cell cycle arrest or apoptosis, flow cytometry were performed. We further examined the expression of cellular proteins of the cell cycle regulating components through Westenblot,. We performed protomic-wide expression analysis using Protein Pathway Array and Ingenuity Pathway Analysis(IPA)software to investigate differences in protein expression between PD0332991 treated and untreated gastric cancer cells.Findings:A total of 22 proteins were differentially expressed between normal and tumor tissues in training set by paired t-test. Of them,9 proteins were selected to build the predictor to classify normal and tumor samples by cross-validation analysis. Ten proteins were differentially expressed among different T stages and 4 of them were able to separate the tumors into 2 main groups:group A represents less invasive tumors and group B more aggressive ones. Additional 4 proteins were found to be associated with lymph node metastasis. Two proteins (AKT and CDK2) were identified as independent risk factors for overall survival along with age and TNM staging.In the second part we found that Embelin inhibits cell growth in gastric cancer cells.We also observed that embelin induced S and G2/M cell cycle arrest, possibly by regulating S phase progression and G2/M checkpoint regulating components such as cdc2, cdc25, and cdk2. In all three cell lines, Protein Pathway Array and IPA analysis identified that the XIAP inhibitor embelin induce a potent alteration of the proteins involved in many cell functions and signal pathways.In the third part,we found that PD0332991 inhibited cell proliferation in human gastric cancer cell lines, the anti-proliferative effect of PD0332991 in gastric cancer cells was related to the ability of PD0332991 to provoke growth inhibition at the G0/G1 arrest, and we also observed that PD0332991 induced G0/G1 arrest through dephosphorylation of Rb at the Cdk4/6-specific site of Ser780. Using large scale proteomic analysis we have identified altered profile of several proteins in PD0332991 exposed gastric cancer cells. The analysis of our data demonstrates that PD0332991 induced significant effect on proteins involved in the regulation of a number of critical tumor processes including cellular development, cell death, cell cycle, cell growth and proliferation, genetic disorder, and cell movement. The proteins regulated by PD0332991 in gastric cancer were classified in several canonical pathways, and the most represented(with the top score by IPA) were molecular mechanism of cancer signaling, p53 signaling, and PI3K/Akt signaling(fig), which have been previously reported to be involved in cancer progression.Conclusion:Our data demonstrated a broad dysregulation of signaling proteins in gastric cancer and these proteins can be selected as clinically useful biomarkers. Different sets of proteins may be used to classify tumor invasion and metastasis and to predict survival in patients with gastric cancers. Embelin induced cell growth inhibition in gastric cancer cell lines and induced S and G2/M cell cycle arrest, possibly by regulating S phase progression and G2/M checkpoint regulating components, PD0332991 induced G0/G1 arrest through dephospho-rylation of Rb at the Cdk4/6-specific site of Ser780,and led to inhibition of cell proliferation. Both of them can induce a potent alteration of the proteins involved in many cell functions and signal pathways.