We performed pathway analysis using Ingenuity Pathway Analysis on the DEGs in both the HCT116 and HT29 cell selleck chemicals Rapamycin lines, treated with LBH589 and vorinostat. In the HCT116 cells, 2289 of the 3043 DEGs and 1679 of the 2232 DEGs in the HT29 cells mapped to defined genetic networks in IPA Knowledge Base. Five networks were found to be altered by HDACi in that they possessed sig nificantly more of the identified DEGs present than would be expected by random chance. These networks included cell cycle. DNA replication, recombination and repair. apoptosis. gene expression and cell growth and prolifera tion. The mapped DEGs were subsequently analyzed for the top 12 canonical biological pathways that demon strated significance within each dataset. In HCT116 cells, 5 common pathways were modulated by both HDACi.

coagulation system, pyrimidine metabolism, metabolism of xenobiotics, arachidonic acid metabolism and fatty acid metabolism. In HT29 cells, 7 common pathways were modulated by both HDACi. arginine and proline metabolism. urea cycle and metabo lism of amino groups. arachidonic acid metabolism. fruc tose and mannose metabolism. pentose phosphotate pathway. nitrogen metabolism and bile acid biosynthesis. Common gene signature of HDAC inhibition in colon cancer cells One of the key objectives of this study was to identify a cassette of genes which were consistently regulated by both vorinostat and LBH589 in both cell lines examined. Such a cassette of consistently regulated genes may serve as molecular markers for HDACi treatment in colon can cer cells.

From the Venn analysis, it is apparent that there is signifi cant differences in how the HCT116 and HT29 cells respond to HDACi treatment. Specifically, when HCT116 and HT29 cells were treated with vorinostat, a combined total of 4688 DEGs were identified. How ever, of this combined total of 4688 DEGs, only 860 genes were transcriptional changes common to both cell lines. Brefeldin_A Similarly, in both cell lines a combined total of 5023 DEGs were identified following treatment with LBH589. How ever, of these 5023 DEGs, only 915 were tran scriptional changes common to both cell lines. From this overlapping gene list, up and down regulated genes in the HCT116 and HT29 cells were directly compared using a 1. 5 fold cutoff. From this com parative list, we identified a panel of 11 genes, 6 genes that are significantly upregulated and 5 that are downregulated in a consistent manner in both cell lines by treatment with both HDACi. Verification of microarray results by quantitative selleck chemical Cisplatin real time RT PCR In order to assess the robustness of the microarray analy sis, quantitative real time RT PCR analysis was performed to validate a selected panel of 15 DEGs and 2 non DEGs, using the primer sets given in Table 4.