ATN-224

Kinase experiment:

Cells are plated in a six-well format (100-300,000 per well) and incubated with ATN-224 at indicated concentrations and for indicated times. Cells are then stimulated with 10 ng/mL FGF-2 for various times and lysed. Lysates are subjected to Western blot analysis using an antibody specific for phosphorylated p44/42 mitogen-activated protein kinase (Thr202/Tyr204) with appropriate signal correction using an antibody specific for p44/42 mitogen-activated protein kinase[1].

Cell experiment:

Human umbilical vein endothelial cells (HUVEC) are maintained in M200/LSGS medium and cells are used between passages 2 and 4 for all experiments. For proliferation assay, cells are plated at 3,000 per well on 0.1% gelatin in M200/2% FBS for 4 hours and then stimulated with 2 ng/mL FGF-2 in the presence or absence of ATN-224 up to 48 hours. HUVEC proliferation is determined using either the Alamar Blue or the MTT assay. Multiple myeloma MM1S cells are grown in RPMI 1640 with 10% fetal bovine serum and 2 mM L-glutamine. HL-60 promyelocytic leukemia cells and MOLT-4 acute myeloblastic leukemia cells are plated at 400,000/mL in T-75 flasks and incubated for 48 to 96 hours for proliferation assays. MMS1 cell proliferation is determined using calcein AM[1].

Animal experiment:

Mice[1] Cold Matrigel (500 μL) is mixed with 800 ng/mL FGF-2 or 300 ng/mL VEGF and heparin (50 μg/mL). Negative control plugs did not contain the proangiogenic factors. The Matrigel mixture is injected s.c. into 4- to 8-week-old female BALB/c nude mice. In some experiments, either ATN-224 (94 μM) with or without Mn-TBAP (100 μM) or water is added directly to the Matrigel plug in the treated and negative control groups, respectively. Alternatively, mice are treated by oral gavage either with distilled water or ATN-224 everyday from Monday to Friday. Animals are sacrificed and the plugs are recovered 5 days after plug injection. The plugs are then minced and homogenized with a tissue homogenizer, and hemoglobin levels in the plugs are determined using Drabkin's solution.

References:

[1]. Juarez JC, et al. Copper binding by tetrathiomolybdate attenuates angiogenesis and tumor cell proliferation through the inhibition of superoxide dismutase 1. Clin Cancer Res. 2006 Aug 15;12(16):4974-82.
[2]. Lin J, et al. A non-comparative randomized phase II study of 2 doses of ATN-224, a copper/zinc superoxide dismutase inhibitor, in patients with biochemically recurrent hormone-naïve prostate cancer. Urol Oncol. 2013 Jul;31(5):581-8.