Impact of CXCR4/CXCL12 axis inhibition in SW480 cell line. (A) Effect of CXCR4/CXCL12 axis inhibition on Akt and Erk phosphorylation. Cells are treated with CXCR4 and CXCR7 siRNAs in hypoxia (1% O2) in presence of 0.5 nM CXCL12 for 15 minutes with or without the CXCR4 antagonist AMD 3100 (10 μM). (B) SW480 cell migration in hypoxia (3% and 1% O2) using a Boyden chamber assay. After 24 h incubation, cells remaining on the upper chamber are mechanically removed. The cells that have migrated to the lower chamber are counted after staining with fluorescent dye (DAPI or 4',6-diamidino-2-phenylindole). Quantification was performed by microscope counting five random fields for each chamber (* p < 0.001). (C) SW480 cell migration was analyzed in presence of CXCR4 or CXCR7 siRNA. There was a significant reduction in migration (37% and 17%, respectively. (D) The effect of increasing doses of chalcone 4 (0.1 μM, 1 μM and 10 μM) was measured on cells migrating under hypoxic conditions. Chalcone 4 at 1 μM and 10 μM reduced migration by 23% and 80%, respectively. (E) Effect of the combination of chalcone 4 and irinotecan at 1 μM. Although irinotecan inhibited migration by 20% (*p <0.01), the irinotecan and chalcone 4 combination further increased the inhibition to 40% (**, p = 0.001).