We hypothesized that even after a transient passage through hypoxia, circu lating tumor cells, although exposed to normoxic condi tions in the bloodstream, will maintain CXCR4 expression at the cell membrane, allowing the metastatic process via the CXCL12 gradient between the primary tumor site selleck chemicals and the liver. Flow cytometry allowed us to analyze CXCR4 expression Inhibitors,Modulators,Libraries in the colon cell lines cultured 24 hours in hypoxia and further maintained for 8, 24 and 48 hours in normoxia. CXCR4 protein level remained elevated at the cell membrane in the three cell lines for 8 to 24 hours. At 48 hours CXCR4 immunoreactivity remained significantly higher in HT29 and SW480 cells. Akt and Erk oncogenic pathways are strongly activated by stimulation of the CXCR4 CXCL12 axis in vitro Modulation by CXCL12 Our aim was to analyze the effect of CXCL12 stimulation on the Akt and Erk oncogenic pathways.

Inhibitors,Modulators,Libraries SW480 cells were stimulated with 0. 5 nM and 50 nM CXCL12. The cells were starved for 4 h in a serum free medium before adding CXCL12, then maintained either in normoxia or in hypoxia and evaluated for protein kinase phos phorylation. In normoxia, both pathways were activated with CXCL12. This activation, however, was weak as compared to that observed in hypoxia at 15 mi nutes, more specifically for the level of Akt phosphoryl ation. Increasing the CXCL12 concentration to 50 nM did not further enhance the level of Akt and Erk phosphorylation. Impact of CXCR4 CXCL12 axis inhibition To study the effect of CXCR4 CXCL12 axis inhibition on Akt and Erk phosphorylation, the SW480 cell line was treated with CXCR4 and CXCR7 siRNAs in hypoxia in the presence of 0.

5 nM CXCL12 for 15 mi nutes. Reducing CXCR4 expression Inhibitors,Modulators,Libraries impaired CXCL12 induced Akt phosphorylation, whereas Inhibitors,Modulators,Libraries Erk activation remained unchanged. Simultaneously blocking the CXCL12 CXCR4 inter action with AMD3100 and inhibiting CXCR4 expression with siRNA fully blocked CXCL12 induced Akt activation, still without affecting Erk activation. Targeting CXCR7 and CXCL12 CXCR7 interaction affected neither Akt nor Erk signaling. Thus, the CXCL12 CXCR4 axis, but not the CXCL12 CXCR7 axis, is able to modulate the Akt pathway. Therapeutic Inhibitors,Modulators,Libraries significance of targeting HIF www.selleckchem.com/products/epz-5676.html 1 and CXCR4 CXCL12 to prevent the dissemination process in vitro The migration of SW480 cells was significantly increased in hypoxia com pared to normoxia. In the presence of CXCR4 or CXCR7 siRNA, a significant reduction in cell migration occurred and 17%, respectively. Treatment with both CXCR4 and CXCR7 siRNAs did not further decrease cell migration. Previous studies showed that irinotecan, a standard chemotherapeutic drug for metastatic colon cancer, has a cytostatic effect on xenografted colon tumors through the inhibition of HIF 1 expression.