AIM: To investigate the effect and mechanism of blockade of the CXC chemokine receptor-4 (CXCR4) signaling pathway by AMD3100 a small non-peptide CXCR4 inhibitor on invasion and metastasis of colorectal cancer cells and βwere constructed on the basis of published sequences. IEC-6). In particular our data showed lymph-node-metastasis-derived cell line SW480 expressed CXCR4 at a high level by using RT-PCR and Western blotting (Figure ?(Figure1).1). We also examined mRNA expression of mRNA in any of the colorectal cancer cell lines (data not shown). Figure 1 Expression of CXCR4 in intestinal epithelial cells. Expression of in highly metastatic CRC cell line SW480 using RT-PCR analysis of mRNA and Western blotting of CXCR4 protein levels. Effect of AMD3100 on viability of CRC cells SW480 In our previous studies we found no expression of mRNA in any of the CRC cancer cell lines. After 3 d incubation greatly enhanced SW480 cells viability in the absence of serum (Figure ?(Figure2).2). The enhancing effect of on cell proliferation was strongly inhibited by treatment with different doses of AMD3100. In a dose-dependent fashion the proliferation rate was decreased to 6.10 ± 0.13 4.49 ± 0.22 3.58 ± 0.13 respectively (< 0.05). The result of 100 and 1000 ng/mL AMD3100 was RO4929097 statistically significant (< 0.01 = 8) in comparison to that of the CXCL12 group (7.97 ± 0.811). Although a reduction in proliferation was also seen in the AMD3100 only group set alongside the serum-free cells (vehicle-treated cells) the inhibition price was not significantly different probably due to a specific effect of blocking CXCL12-CXCR4 interaction. The assay also revealed that in 24 h there was no significant difference in viability in any of the groups. Therefore the cell invasion assay was performed at 24 h to remove its influence on cell viability. 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 ... Effect of AMD3100 on invasion of CRC cells To evaluate the effects of inhibition of CXCL12-CXCR4 interaction on CRC invasion we performed an invasion assay using AMD3100. After 24 h incubation AMD3100 markedly reduced invasion of SW480 cells at concentrations of 100 Rabbit Polyclonal to Connexin 43 (phospho-Ser265). and 1000 ng/mL (Table ?(Table1) 1 by 28.43% (< 0.05) and 77.23% (< 0.01) respectively. Table 1 Effect of AMD3100 on invasion of CRC cells (mean ± SD) Effect of AMD3100 on chemotactic migration of CRC cells The effect of AMD3100 on inhibiting RO4929097 CXCL12-induced migration of CRC cells was estimated by a classical chemotaxis assay. The selected CXCR4-positive cell line SW480 did migrate in response to CXCL12 in a classical chemotaxis assay with an optimal response at 100 ng/mL. After AMD3100 treatment chemotactic activity of SW480 cells was reduced in a dose-dependent manner (Figure ?(Figure3B).3B). The inhibition rate with AMD3100 at 10 100 and 1000 ng/mL was 5.24% 47.27% and 62.37% respectively. The latter two achieved a significant difference compare to the control group (a b and c in Figure ?Figure3A3A). 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 RO4929097 SW480 cells at final concentrations of 100 and 1000 ng/mL. The blue-stained cells are those ... Effect of AMD3100 on expression of MMP-2 MMP-9 and VEGF in SW480 cells The CXCL12-CXCR4 axis contributes to invasion and specific organ metastasis through regulation of RO4929097 its target genes which have recently been shown to be and but not and mRNAs in SW480 cells was significantly downregulated by 100 and 1000 ng/mL AMD3100. Densitometric analysis revealed the relative expression reduced to 17.58% ± 3.79% for < 0.05). Shape 4 A: Aftereffect of AMD3100 on manifestation of MMP-2 VEGF and MMP-9 in SW480 cells. Protein examples extracted from SW480 cells treated for 26 h with AMD3100 had been subjected to Traditional western blotting for MMP-2 MMP-9 VEGF and GAPDH protein. AMD3100 decreased significantly ... DISCUSSION An evergrowing body of books offers indicated CXCR4 can be important in a number of malignancies and more particularly that receptor could be a propitious focus on in treating tumor. In experimental systems convincing proof shows that selective inhibition of CXCR4 suppresses CXCL12-induced migration of tumor cells invasion neoangiogenesis and metastases. Neutralizing the relationships of CXCL12 and CXCR4 by monoclonal antibody significantly impairs metastasis of breast cancer cells to regional lymph nodes and lungs[27]. Human breast tumor.