Unless otherwise indicated, the results are expressed as the meanSD from at least three independent experiments. MO, USA). TPC-1 cells were grown in DMEM (Gibco) supplemented with 10% FBS. Baf3 cells were grown in RPMI-1640 (Gibco) supplemented with 10% NBSC (Gibco) and IL3. Baf3 cells transfected with RET were grown in RPMI-1640 (Gibco) supplemented with 10% FBS and 2 g/mL puromycin. Proliferation assays Thyroid cancer cells were seeded in 96-well plates at an appropriate density of 3000-10 000 cells per well and cultured for 24 h before exposure to increasing doses of AL3810. After being cultured for 3 or 5 d, TT, TPC-1 and SW579 cells were fixed with 10% trichloroacetic acid (TCA) and stained with SRB (Sigma) in 1% acetic acid (cell death detection Kit-POD reagent (Roche, Basel, Switzerland) at 4 C overnight. After being washed with PBS, the slides were incubated with DAKO secondary antibody (EnVision? Detection System, DAKO), and peroxidase activity was detected with diaminobenzidine (DAB). Three random areas from three individually stained tumors were captured with a Leica Upright Metallurgical Microscope (Leica Microsystems, Wetzlar, Germany). Tumor xenograft assays Athymic BALB/c nude mice aged 4C6 weeks were housed and maintained under specific pathogen-free conditions with a 12 h light/dark cycle at 251 C and received food and water were calculated as follows: test was applied for statistical comparisons using GraphPad Prism 6. Unless otherwise indicated, the results are expressed as the meanSD from at least three independent experiments. Differences were considered to be significant at and compared to XL184, ZD6474 and Sorafenib. Clinical trials of multikinase inhibitors that target vascular endothelial growth factor (VEGF) receptor signaling have shown efficacy in thyroid cancer30,31. Thyroid cells require contact with capillaries to function normally and secrete trophic signals for capillary endothelial cells, primarily VEGF32. On Rabbit polyclonal to MMP1 transformation and loss of polarity, a disorganized tumor vasculature may result in cancer-cell hypoxia, a loss of immune surveillance, increased VEGF receptor activation, and a dependence on VEGFR signaling that can be leveraged therapeutically33. Accordingly, the administration of AL3810 significantly reduced the growth of xenografts and efficiently inhibited the activation of endothelial cell-expressed VEGFR2 and PDGFR tyrosine kinases, which decreased tumor microvessel density. The magnitude of the antiangiogenic effect of AL3810 suggests that its overall antitumor effects may be due to its antiangiogenic properties. RET is a well-defined clinical target in thyroid cancer, as recently demonstrated by XL184 and ZD6474, two multikinase inhibitors that are approved for the treatment of advanced MTC34,35,36,37. Although AL3810 inhibits RET kinase activity at submicromolar concentrations in enzymatic assays, the high potency shown by this compound against RET-dependent Baf3 KRas G12C inhibitor 4 cells was comparable to its activity against RET-negative cells, with at least 10-fold selectivity KRas G12C inhibitor 4 versus Baf3 parent cells. These results demonstrate that the inhibition activity in RET may contribute to antitumor activity of AL3810. In conclusion, AL3810 showed promising antitumor activity against human thyroid cancer, which was due to its inhibition KRas G12C inhibitor 4 of angiogenesis. In addition, the AL3810-mediated inhibition of RET may also inhibit thyroid tumors harboring RET alterations. These data suggest that AL3810 is an effective therapeutic agent in thyroid cancer that warrants further clinical development. Acknowledgments This work was supported by the Personalized Medicines-Molecular Signature-based Drug Discovery and Development, Strategic Priority Research Program of the Chinese.
