Immunoprecipitation and immunoblotting selleck chem Cells were lysed in cold RIPA buffer supplemented with a proteinase inhibitor cocktail and a phosphatase inhibitor cocktail. Protein concen tration was determined using a detergent compatible protein assay according to the manufacturers instruc tions. Protein from each sample was immunoprecipitated overnight at 4 C with an anti VEGFR 2, anti PDGFR B, or anti FGFR 1 antibody plus protein GA agarose beads. Immune complexes were washed with cold RIPA buffer containing proteinase inhibitors and phosphatase inhibitor. Proteins were eluted by boiling in Inhibitors,Modulators,Libraries SDS sample buffer, separated by SDS PAGE, and transferred to polyvi nylidene difluoride membrane. Membranes were probed with an anti phosphotyrosine antibody and then stripped with stripping buffer.

To detect total Inhibitors,Modulators,Libraries VEGFR 2, PDGFR B, and FGFR 1 levels, membranes were re probed Inhibitors,Modulators,Libraries with the same anti VEGFR 2, anti PDGFR B, and anti FGFR 1 antibody that was used for the immunoprecipitation. Immunoblot ting of phospho ERK12 and ERK12 was performed on whole cell lysates with B actin as a loading control. Statistical analysis Continuous data were expressed as median and range and were compared between groups using one way ANOVA and Dunnett t test. Categorical variables were compared using the chi square test, or Fishers exact test, where appropriate. All data were analyzed using the SPSS 13. 0 computer program, and significant difference was defined as P 0. 05. Results Dovitinib inhibited HCC xenograft tumor growth and metastasis The therapeutic effect of dovitinib was examined using the orthotopic HCC model.

Continuous dovitinib treat ment for 2 weeks at doses of 25 or 50 mgkg was started Inhibitors,Modulators,Libraries 14 days after orthotopic injection of MHCC 97H, SMMC7721, or QGY 7703 cells. There was no signifi cant change in body weight of the animals in each treat ment group compared with that of the control animals. Dovitinib significantly inhibited pri mary tumor growth in a dose dependent manner relative to the control group. In MHCC 97H, a HCC cell line with high metastasis capability, doviti nib also Inhibitors,Modulators,Libraries inhibited pulmonary metastasis in a dose dependent manner. Dovitinib inhibited RTK signaling pathways in vitro Pharmacokinetic and pharmacodynamic studies have revealed that the pharmacologically relevant concentra tion of dovitinib is 0. 010. 3 umolL.

To evalu ate the potential effect of dovitinib at pharmacological concentration on the activation of RTK signaling path ways in vitro, we first examined the expression and ac tivation of VEGFR, under FGFR, PDGFR, Flt 3, and c KIT in HCC cell lines and endothelial cell lines by immuno blotting. FGFR 3 was expressed by Hep3B and MHCC 97H, VEGFR 1 was expressed by two endothelial cells, PDGFR B was clearly expressed by two cell lines, MHCC 97H and SMMC7721. In contrast, four of the five endothelial cell lines homogenously expressed VEGFR 2 and FGFR 1. Flt 3 and c KIT were undetectable in all cell lines.