The antitumor effect against A431 xenografts is smaller than previous TKIs because we used less frequent administration to identify a dose schedule suitable for use with chemotherapy. of AG1478 significantly AM 103 enhanced the effectiveness of cytotoxic medicines, with the combination of AG1478 and temozolomide showing synergistic antitumor activity against human Mouse monoclonal to UBE1L being glioma xenografts. AG1478 was also examined in combination with mAb 806, an anti-EGFR antibody that was raised against the de2C7 EGFR but unexpectedly also binds a subset of the EGFR indicated in cells exhibiting amplification of the gene. The combination of AG1478 and mAb 806 displayed AM 103 additive, and in some cases synergistic, antitumor activity against tumor xenografts overexpressing the EGFR. Here, we demonstrate that different classes of inhibitors to the EGFR can have synergistic antitumor activity mitogenic assay by using IL-3-dependent BaF/3 cells transfected with the WT EGFR (19). For animal experiments, the AG1478 was dissolved in 100 mM Captisol (Cydex, Overland Park, KS) at the desired concentration. The concentration of AG1478 in serum and cells was essentially performed as previously validated (20). Fluorescence-Activated Cell Sorter Analysis of EGFR Manifestation. Cells were incubated in serum free media overnight and then incubated with new media comprising AG1478 or EGF for 10 or 240 min. Cells were then incubated with mAb 806 for 30 min at 4C with bound antibody detected by using an FITC-coupled goat anti-mouse antibody (Calbiochem). Cells were analyzed on an Epics Elite ESP circulation cytometer (Beckman Coulter) and analyzed by using expo for windows. ELISA Analysis of EGFR Manifestation. A431 cells were incubated over night in serum-free press and then incubated with new press comprising AG1478 for 10 min. Cells were placed in lysis buffer (1% Triton X-100/30 mM Hepes/150 mM NaCl/500 M 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF)/150 nM aprotinin/1 M E-64 protease inhibitor/0.5 mM EDTA/1 M leupeptin, pH 7.4) for 1 h at 4C. Lysates were clarified by centrifugation and diluted with AM 103 PBS, and the EGFR was assayed by ELISA as explained (21). Antiproliferative Assays. A431 and U87MG.2C7 cells were setup at a density of 2.5 103 cells per well in 96-well plates, allowed to adhere overnight, and then incubated with AG1478 or mAb 806 for 48 h. After 0 and 48 h, viable cell number was determined by using the MTS assay (Promega), and the percentage inhibition was determined by the following method: 1 – [A490 (48 h) of treated cells – A490 AM 103 (0 h)]/[A490 (48 h) of control cells – A490 (0 h)] 100. Xenograft Models. A431 or U87MG.2C7 tumor cells were inoculated s.c. into both flanks of woman BALB/c nu/nu mice. Because of variations in xenograft growth rate, mice were constantly inoculated with the same cells on each flank. The therapeutic effectiveness of AG1478 only or in combination was investigated in both preventative and founded tumor models as explained (10). Variations between treatment organizations at given time points were tested for statistical significance by using Student’s test. Immunohistochemistry of Xenografts. Xenografts were inlayed in OCT compound (Sakura Finetek, Torrance, CA) and snap freezing. Sections were slice, fixed in acetone for 10 min, and stained with antibodies to the EGFR (sc-03), phosphorylated EGFR (tyrosine 1173), and phosphorylated Akt (serine 473), all purchased from Santa Cruz Biotechnology. Results Biodistribution of Soluble AG1478. We have previously shown that serum levels of soluble AG1478 peaked 30 min after s.c. administration (20). Accordingly, the level of AG1478 in normal cells and U87MG. 2C7 xenografts was identified at this time point. Serum AG1478 levels were proportional to dose and consistent between mice, having a mean SD concentration of 23 5 M observed 30 min after a 400-g i.p. injection and 59 12 M after a 1-mg injection (Fig. 1). The.
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