In vitroactivity: Reversine induces myogenic lineage-committed cells to become multipotent mesenchymal progenitor cells, which proliferates and redifferentiates into bone and fat cells. Reversine, as an A3 adenosine receptor antagonist, competitively inhibits forskolin-stimulated cAMP production in stably transfected Chinese hamster ovary (CHO) cells. Reversine inhibits the phosphorylation of a well-known Aurora target, histone H3 in HCT116 cells. Moreover, Reversine potently blocks proliferation of multiple tumor cell types, and induces cell death. In primary human tumor samples, Reversine also inhibits colony formation of leukemic cells. When treated in combination, reversine and aspirin synergistically inhibit growth of cervical cancer cells and induce cell apoptosis.

Kinase Assay: Each tube in the A3 AR competitive binding assay contains 100 μL of membrane suspension (20 μg of protein), 50 μL of [125I]4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide (0.5 nM), and 50 μL of increasing concentrations of the test ligands in Tris-HCl buffer (50 mM, pH 7.4) containing 10 mM MgCl2 and 1 mM EDTA. Nonspecific binding is determined using 10 mM 5′-N-ethylcarboxamidoadenosine in the buffer. The mixtures are incubated at 25°C for 60 min. Binding reactions are terminated by filtration through Whatman GF/B filters under reduced pressure using a MT-24 cell harvester. Filters are washed three times with 9 mL of ice-cold buffer. Radioactivity is determined using a Beckman γ-counter, and the percent inhibition is calculated.

Cell Assay: Cell viability of different tumor cell lines (HL-60, A375, HeLa, HCT-116, T47D, and MCF-7 cell lines) is assessed using ATPlite 1step. Briefly, 2 × 104 cells for each well are plated in a 96-well plate in presence of crescent quantity of reversine. After 72 h, the plates are recovered and 100 μL ATPlite solution is added to each well. The plates are shaken for 2 min at 700 rpm and luminescence is measured using EnVision Multilabel plate reader. Each sample is analyzed in triplicate.

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