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T the future clinical evaluation of this compound in colorectal tumors.Offered the truth that choice of anti-EGFR therapies is determined by the presence of K-RAS mutations and that tumors with constitutive activation of downstream mediators can present secondary activating loops, we interrogated if differences in the kinase profile among the two groups could be identified. For that reason, we compared the kinase profile in K-RAS mutated (n = 8) versus non-mutated (n = 10) tumors. Expression of EGFR was equivalent in both groups, but ALK, AKT/Thr308 and STAT1 have been reduced in tumors with K-RAS mutations (Figure 1C). No differences had been observed for the expression of pErk1/2. Other kinases whose phosphorylation was decreased in K-RAS mutated tumors integrated MSPR, FGFR3 and ErbB3 (Figure 1C). Finally, we observed that an important quantity of proteins had been phosphorylated within exactly the same tumor (Figure 1D), supporting the concept that targeting of quite a few proteins or crucial signalling nodes might be a rational approach.Pharmacologic evaluation with multi-kinase inhibitorsNext, we decided to evaluate the effect on cell proliferation of a number of kinase inhibitors developed against by far the most frequently phosphorylated kinases observed in human samples. We evaluated six various agents, which includes some agents approved in cancer for other indications and a multikinase inhibitor presently in 3-Furanoic acid Data Sheet preclinical development. The agents included lapatinib, as an EGFR and ErbB2 inhibitor, sunitinib as a VEGFR2 and PDGFR inhibitor, crizotinib as a c-MET and ALK inhibitor, dasatinib as a Abl, SRC and c-Kit inhibitor, BEZ235 as a dual pan-PI3K/mTOR inhibitor, and NVP-BSK805 as a JAK/STAT inhibitor (Figure 2A). In addition, we evaluated a novel polypharmacology kinase inhibitor termed EC-70124, a hybrid indolocarbazole obtained by combinatorial biosynthesis of Rebeccamycin and Staurosporin genes [10]. The impact on cell proliferation of these compounds was evaluated in two colon cancer cell lines SW620, and HT29 working with the MTT metabolization assay. By carrying out a dose response curve we observed various sensitivity to the drugs evaluated. The proliferation assays showed that the new multi-kinase inhibitor EC-70124 had a robust effect in the cell lines studied compared with other agents. EC-70124 reached a half-maximal inhibitory effect within the nanomolar range (below 200 nM) in the two cell lines (Figure 2A, 2B). At doses below 500 nM only BEZ235 showed a relevant impact on development inhibition in SW620, but restricted in HT29. Dasatinib showed only antiproliferative effect in HT29. We also investigated the impact of EC-70124 in threedimensional growth applying exactly the same cell lines. For this objective, we grew cells in matrigel, a semisolid media where the cells grow forming spherical structures. Treatment with EC-70124 strongly decreased the diameter of these spheres (manage vs remedy, imply diameter and SD = three.62 +/- 0.11 vs two.28 +/- 0.08 and ten,63 +/- 0.7 vs 1.1 +/- 0.1 for SW620 and HT-29, respectively) (Figure 2C).31273 OncotargetRESULTSPhospho-kinase profile of human colorectal tumorsWe analyzed the activation status of several RTKs and relevant signaling mediators in samples from eighteen individuals diagnosed with colorectal cancer. To complete so, we applied two antibody-based array kits that evaluate the phosphorylation status of those proteins, as shown in Supplementary Figure S1. Patient qualities are described in Table 1. The analyses revealed that in the fifty-nine proteins evaluated, only twenty.

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