We hypothesized that irinotecan would inhibit CXCR4 expression selleck bio by inhibiting HIF 1. Chalcone 4 is a neutraligand of CXCL12 and impairs CXCR4 CXCR7 CXCL12 interaction. In addition, other studies have Inhibitors,Modulators,Libraries already shown that inhibition of CXCR4 in vivo inhibits the metastatic process and the migration of breast cancer cells. We have shown that the combination of the two drugs is more effective than each drug separ ately as migration was decreased by more than 40%. Conclusion We have demonstrated for the first time the potential therapeutic significance of inhibiting CXCR4 signaling through a combinatorial approach inhibiting HIF 1 and CXCR4 CXCL12 interaction. CXCR4 seems to be a rele vant target, as CXCR4 remains continuously expressed when tumor cells switch from a hypoxic to a normoxic environment.

Finally, CXCR7 is differentially expressed compared to CXCR4 and could be involved in some subtypes of more aggressive tumors. Thus, Inhibitors,Modulators,Libraries CXCR4 and CXCR7 seem to play different roles in colon tumors, and further studies are necessary to better understand their re spective roles. Materials and methods Tumor specimens Human tumor specimens were obtained at the Gastro intestinal Surgical Department of the University Hospital Hautepierre according to the French Ethical Committee recommendations and the ethical standards of the 1964 Declaration of Helsinki. All pa tients provided written informed consent. Cell culture and treatments Human colon carcinoma HCT 116, HT 29 and SW480 cells were maintained at 37 C under normoxic and hypoxic conditions in DMEM supplemented with 10% fetal bovine serum.

The cells were treated Inhibitors,Modulators,Libraries during expo nential growth conditions. Irinotecan was used at a concentration of 1 uM. AMD3100, an antagonist of CXCR4, was used at 10 uM. Chalcone 4 was provided by JL Galzi. . Migration tests Boyden chambers were used for the in vitro migration assay. The upper and lower compart ments were filled with 1% FCS and 10% FCS, Inhibitors,Modulators,Libraries respectively. Cells were added in the upper compartment. After 24 h, the cells were fixed with 4% paraformaldehyde for 15 min and stained with DAPI. Migrating cells were counted using an epifluorescence microscope. Relative quantitative PCR The mRNA expression of CXCR4, CXCR7, HIF 1 and control PDGF genes was evaluated by relative quantita tive real time PCR analysis using the Light Cycler system and FastStart DNA Master Mix SYBR Green I.

RNA was extracted with Trizol reagent according to the manufacturers protocol. Reverse tran scription of 2 ug RNA was performed using reverse transcriptase and oligo primers. PCR was performed as follows Inhibitors,Modulators,Libraries denaturation at 95 C for 10 min, fol lowed by 40 cycles at 95 C for 20 s and selleckchem 62 C for 20 s and elongation at 72 C for 20 s using the maximum temper ature transition rate of 20 C s. Fluorescence measurements were taken at the end of the elongation phase.