None of the genes had been downregulated in response to treatment. A significant correlation was observed involving the fold-changes in gene expression observed in hypoxia- versus DMOG-treated Caco-2 cells Spearman correlation co-efficient 0.50, p < Inhibitors,Modulators,Libraries 0.001, not shown highlighting the high degree of concordance between hypoxia- and DMOG-mediated responses in Caco-2 CRC cells. The genes whose expression changed the most dramati- cally in response to hypoxia and DMOG were ANGPTL4, EFNA3, TGFβ1 and VEGF. To determine their require- ment for HIF isoforms, a small interfering si RNA approach was used.

Distinct knockdown of HIF-1α and HIF-2α, which we’ve got previously demonstrated in other cell varieties to markedly lower HIF mRNA and protein [38,39], was confirmed in Caco-2 on the mRNA level in each DMOG- and hypoxia-stimulated cells, with 81% and 85% knockdown of HIF-1α mRNA in the veliparib clinical trial presence of siRNA against HIF-1α compared with siLuc-transfected Caco-2 cells and 93% and 86% knockdown of HIF-2α mRNA within the presence of siRNA against HIF-2α information not shown. There was no inhibitory effect of siHIF-1α on HIF-2α, and vice versa data not shown. Certain knockdown of HIF-1α and HIF-2α was also observed with the protein level in cells exposed to hypoxia Figure 2e and DMOG Figure 3e. Expression of ANGPTL4 was dependent on HIF-1α in Caco-2 cells stimulated with both hypoxia or DMOG Figures 2a and 3a with reductions of 83% relative to siLuc-transfected cells, p < 0.001 and 60% p < 0.001 respectively. In contrast, knockdown of HIF-2α was without effect.

Comparable information had been observed for the other genes in cells exposed to hypoxia, with knockdown of HIF-1α, but not of HIF-2α, having a substantial in- hibitory result. Consequently for EFNA3, reductions of 54% p < 0.001, Figure 2b and 43% p < 0.05, Figure 3b kinase inhibitor library for screening were observed in response to hypoxia and DMOG res- pectively in the presence of siHIF-1α. For TGFβ1, reduc- tions of 60% p < 0.001, Figure 2c and 80% p < 0.001, Figure 3c were observed in response to hypoxia and DMOG respectively. Finally, in the case of VEGF, HIF-1α knockdown resulted in reductions of 54% p < 0.001, Figure 2d and 75% p < 0.001, Figure 3d in response to hypoxia and DMOG respectively. These findings suggest that HIF-1, but not HIF-2, mediates the induction of angiogenic genes in CRC cells downstream of HIF activa- tion in response to ether hypoxia or the hypoxia mimetic DMOG.

Examination of Caco-2 responses to EGF alone and in blend with all the hypoxia mimetic DMOG Given that we established that angiogenic gene induction was HIF dependent in Caco-2 cells, we up coming investigated the effect of EGF, alone or in mixture using the hypoxia mimetic agent DMOG, on activation with the HIF pathway in Caco-2 cells. HIF-1α Figure 4a and HIF-2α Figure 4b mRNA decreased modestly following stimulation with both EGF, DMOG or maybe a blend of both EGF and DMOG stimulation, but these variations in level of mRNA across all three groups above a time period of 24 hours weren’t statistically major. In contrast, Western blot analysis demonstrated a consistent up-regulation of each HIF-1α and HIF-2α protein following DMOG or EGF stimulation alone and in blend Figure 4c.