Edited by cell growth and reduced spontaneous apoptosis of CRC cells (16). Closing Lich has been suggested that autocrine signaling VEGF/VEGFR1 survive in synergy with the EGFR by the tumor cells f Rdern and / or distribute an important therapeutic significance of these findings is that strategies to block VEGF or EGFR signaling inhibition of extracellular Ren ligands or receptors, as is the case for monoclonal antibody body, a portion to prevent the oncogenic signaling. However, we expect that small molecule TKI confinement be able to k Nnte interference in the internal RTK signaling and cross-talk Lich VEGF/VEGFR1 intracrine loop. To test this hypothesis, w We hlten Including two TKI Lich vargatef / BIBF 1120, an inhibitor angiokinase Triple VEGFR, PDGFR and FGFR (19), and afatinib / BIBW 2992, the irreversibly inhibits EGFR and HER2 (20). Vargatef is currently in phase III trials in non-small cell lung cancer Center (NSCLC) and ovarian cancer, w Has afatinib during phase III trials for the treatment of NSCLC and breast cancer reached. We now report that afatinib vargatef a synergistic activity and all t in models of CRC, which are refractory R on the combination of bevacizumab and cetuximab and show the mechanistic differences between ITC and monoclonal antibodies Rpern aufzukl Ren. In particular, our results show that TKI only able to reduce intracellular Re-EGFR and VEGFR signaling, which is accompanied by induction of cell death by apoptosis. Our results provide a mechanistic explanation Tion for the failure of monoclonal antibodies Rpern and show that EGFR and VEGF rational weight Hlt (R)-targeted agents are combined with a clinical benefit nnten k. The antitumor effect of targeted molecular agents were in athymic M Mice (NMRI Foxn1n female, aged 6 weeks) evaluated from Taconic Flavopiridol 131740-09-5 bearing HT-29 CRC xenografts. Two million cells were injected into the right flank, and began the treatment, tumors were felt as (average size E of the tumor 100 mm3). The animals were t Resembled weighed and the tumor size E was estimated three times a week. The tumor volume (mm3) were calculated using the formula: [2 ( Width length2) /]. Boxplot analysis of the weights and tumor volume was measured with GraphPad Prism version 5.00 (GraphPad Software). Treated / contr values were calculated as follows: (average tumor volume of treated animals / mean tumor volume of animals controlled On) 100th The animals were treated in accordance with institutional guidelines. All analyzes were performed with the aid of biomarkers from purchase Flavopiridol tumors after 18 days of drug exposure with minimal necrosis collected. Administered intraperitoneally 48 hours before the victim was (500 mg), and the DNA synthesis in vivo, the thymidine analogue 5-ethynyl-20-deoxyuridine (EDU Invitrogen) to measure. The incorporated EdU was by a coupling reaction fluorescent azide (Click-iT, Invitrogen) from paraffin tumor samples and

AC220 disadvantages diamidino-40-0.6-2-phenylindole revealed in order to reveal the nuclei of individual cells. The percentage of apoptotic tumor cells was supported by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (in the in situ Cell Death Detection Kit, Roche Applied Science) Hat. The following antique body were used for immunohistochemical analysis: anti-phospho-EGFR (# 12351, Santa Cruz), recognize the Tyr1173-phosphorylated EGFR, anti-phospho-VEGFR1 antibody antibodies (# 07-758, Millipore), the Tyr1213-phosphorylated VEGFR1 recognize, and the states ndigen points Cy3-conjugated secondary Ren Antique rpern (Jackson ImmunoResearch). All images were acquired with a fluorescence microscope and fluorescence Th were measured using MetaMorph software (Universal Imaging Corporation) for the quantitative analysis. Repr for the quantitative analysis of DNA synthesis in vivo (installation EDU), the data The context sentieren between education and the positive cells and the total number of living cells and are mean values from five fields per tumor (each field is approximately 1,700 cells) from three different tumors. For the quantitative determination of apoptosis,

the data represent the ratio Ratio of TUNEL-positive apoptotic cells and total viable cells and are mean values of five fields per tumor of four tumors. For the quantitative Flavopiridol Alvocidib analysis of the intensity t of the signal for phospho-EGFR and phospho-VEGFR1, repr sentieren Data, the mean fluorescence intensity t of treated tumors, compared with the intensity of t the treatment of tumors controlled And are averages from five fields per tumor from four different tumors. The tumor cells were kindly provided by Richard Camalier (Division of Cancer Treatment and Diagnosis Tumor Repository, National Cancer Institute) and Richard Hamelin (Paris, France) provided. The ability Lebensf Of the cells was determined by the MTT Viabilit Tstest determined after 120 hours of continuous exposure to drugs, as previously described (21). Cell cycle analysis was performed as described (22), w While the percentage of apoptotic cells by flow cytometry with the APO-BrdU kit from BD Biosciences was expertized gt. Combination drug reactions who