[PubMed] [Google Scholar] 26. resistance, we measured the amount of HGF in the CM from fibroblasts. HGF is not expressed in epithelial cells and therefore c-Met receptor requires HGF production by surrounding stromal cells for ligand-dependent activation Clofarabine [37]. As shown in Figure ?Determine2A,2A, the ELISA assay demonstrated that HGF production is only shown in CM from fibroblast. The c-Met receptor tyrosine kinase activation induces pleiotropic biological effects in a wide variety of cells, including mitogenic, motogenic, morphogenic, and anti-apoptotic activities [10, 38]. To determine whether fibroblast-derived HGF activates the c-Met receptor resulting in CPT-11 resistance, we treated cells with CM from fibroblasts. We detected c-Met activation by treatment of fibroblasts-derived CM (Physique ?(Figure2B).2B). To mimic the tumor microenvironment, we performed co-culture experiments with fibroblasts and cancer cells. We investigated whether fibroblasts in co-culture adopted compensatory mechanisms such as the activation of the c-Met receptor and increased resistance to CPT-11 by cancer cells. As shown in Figure ?Physique2C2C and ?and2D,2D, co-culture with fibroblasts induced resistance to CPT-11 and activated the c-Met receptor in cancer cells. To determine whether HGF is usually directly implicated in the activation of c-Met and the resistance to Rabbit Polyclonal to MDM2 (phospho-Ser166) CPT-11, we knocked-down HGF by siRNA and measured cell viability in the presence of CPT-11. The HGF siRNA significantly Clofarabine suppressed HGF expression in CCD-18co cells (Physique ?(Figure2E).2E). As expected, CM from HGF siRNA treated cells failed to rescue malignancy cells from the apoptosis by CPT-11 (Physique ?(Figure2F).2F). These results confirm the importance of fibroblast-derived HGF in CPT-11 resistance of cancer cells and indicate that HGF might be a therapeutic target for overcoming resistance to CPT-11. Open in a separate window Physique 2 fibroblast-derived HGF Clofarabine activates c-MET receptor and induces CPT-11 resistance in colorectal cancer cellA. HGF secreted by cancer cells (HCT-116 and DLD-1) and colonic fibroblasts (CCD-18co) were measured. Cells were cultured with serum free medium for 24 h and HGF concentrations were determined by ELISA. B. CM from fibroblast activates c-MET receptors. HCT-116 and DLD-1 cells were cultured with serum free media or CCD-18co CM for 1 h. Cells were collected, and the indicated proteins were detected by western blotting. C. Colonic fibroblast cells promote CPT-11 resistance of colorectal cancer cells (HCT-116 and DLD-1). Cancer cells were cultured with (white bar) or without (black bar) CCD-18co cells, in the presence or absence of CPT-11 (1.25-20 M) for 48 h, and inhibition of cell proliferation was determined by MTT assay. Significant differences were evaluated using an unpaired two-tailed Student’s < 0.05 and ***< 0.001). D. Co-culture with colonic fibroblast CCD-18co cells increases c-MET receptor activation in colorectal cancer cells. HCT-116 and DLD-1 cells were co-cultured with CCD-18co cells for 24 h. Lysates were analyzed for c-MET activation by western blotting. E. Inhibition of HGF production from fibroblast by transfection with HGF siRNA. Colonic fibroblast cells were transfected with 10 nM HGF siRNA or scramble siRNA. After transfection, cells were collected and lysates were submitted to Western blotting to quantify HGF. F. HCT-116 and DLD-1 cells were cultured with CM from HGF siRNA transfected fibroblast for 48 h in the presence or absent of CPT-11. Cell viability was determined by MTT assay. Significant differences were evaluated using an unpaired two-tailed Student's < 0.05, **< 0.01 and ***< 0.001). Targeting of fibroblast-derived HGF abrogates HGF stimulated CPT-11 resistance in colorectal cancer cells To test whether HGF directly contributes to the effect on overcoming CPT-11 resistance in cancer cells, anti-HGF antibody was added to the CM to neutralize the HGF activity. The viability of cancer cells in the CM from fibroblasts was significantly decreased by the addition of 200 ng/ml of the anti-HGF antibody (Determine ?(Figure3A).3A). To determine the inhibitory effect of anti-HGF antibody in conditions mimicking a tumor microenvironment, co-cultures of fibroblasts and cancer cells were Clofarabine subjected to MTT assay (Physique ?(Figure3B).3B). As shown in Figure ?Physique3B,3B, neutralization of fibroblast-derived HGF maintained the reduction in cell viability induced by CPT-11 indicating that HGF targeting significantly enhances CPT-11 stimulated anti-cancer activity. Taken together, our findings suggest.