Inhibition of CXCR4 activity with AMD3100 decreases invasion of human colorectal cancer cells in vitro

Figure 1 Expression of CXCR4 in intestinal epithelial cells. Expression of CXCR4 in highly metastatic CRC cell line SW480, using RT-PCR analysis of mRNA, and Western blotting of CXCR4 protein levels.

Figure 2 Effect of AMD3100 on viability of CRC SW480 cells. After 24 h incubation, cells growing in 96-well plates were treated with AMD3100 for 2 h. CXCL12 was added at 20 ng/mL per day, and the MTT assay revealed that in serum-free medium or the absence of CXCL12, AMD3100-induced inhibition was relatively weak. CXCL12-induced cell proliferation was significantly suppressed by 100 and 1000 ng/mL AMD3100 in SW480 cells. Cell viability was not significantly affected by 10 ng/mL AMD3100 (compared to the unstimulated group). Data are mean ± SD of eight wells after 3 d incubation. Bars indicate mean ± SD of triplicate experiments.

Figure 3 A: Effect of AMD3100 on chemotactic migration of CRC cells. The chemotaxis assay indicated that AMD3100 significantly inhibited the CXCL12-mediated migration of SW480 cells at final concentrations of 100 and 1000 ng/mL. The blue-stained cells are those that migrated through the polycarbonate membrane to the lower surface of the membrane (a-c); B: CXCL12 inhibited migration of SW480 cells in a dose-dependent manner. Bars indicate mean ± SD of triplicate experiments. ^aP < 0.05, ^bP < 0.01.

Figure 4 A: Effect of AMD3100 on expression of MMP-2, MMP-9 and VEGF in SW480 cells. Protein samples extracted from SW480 cells treated for 26 h with AMD3100 were subjected to Western blotting for MMP-2, MMP-9, VEGF and GAPDH proteins. AMD3100 significantly decreased MMP-9 and VEGF protein expression in SW480 cells in a dose-dependent manner. The level of GAPDH expression was determined as a control for equivalent protein loading; B: RNA samples extracted from SW480 cells treated for 26 h with AMD3100 were subjected to RT-PCR for MMP-2, MMP-9, VEGF and β-actin. AMD3100 also significantly decreased expression of MMP-9 and VEGF mRNAs in SW480 cells, and the inhibitory effect was dose-dependent. RT-PCR for β-actin was performed in parallel to show an equal amount of total RNA in the sample.