Novel Application of Radotinib for the Treatment of Solid Tumors via Natural Killer Cell Activation

Figure 2

Radotinib enhances cytolytic activity of NK cells against Fas-expressing A549 cells. (a) Primary NK cells were isolated from healthy donors to perform NK cell cytotoxicity assay. The purity of CD3^-CD56⁺ NK cells was 96.6%. (b) To determine the effect of radotinib on the cytolytic activity of NK cells against A549 cells, the cells were treated with various concentrations of radotinib (0, 12.5, 25, 50, 100, and 200 μM) for 48 h and the cytotoxicity assay was performed (E : T  : 1). (c) To determine if the effect of radotinib on NK cytotoxicity was mediated by the Fas receptor, Fas expression was transiently downregulated by Fas siRNA transfection into A549 cells. At approximately 70% confluency, A549 cells were incubated with 50 pmole Fas-specific siRNA or negative control siRNA using Lipofectamine RNAiMAX. Surface expression of Fas on A549 cells was determined by staining with PE-conjugated Fas antibody (solid line). PE-conjugated mouse IgG antibody was used as an isotype control (dotted line). (d) The effect of radotinib on NK cytolytic activity against Fas siRNA-transfected A549 cells was determined by cytotoxicity assay. Radotinib-treated NK cells were used as effector cells, and Fas siRNA-transfected A549 cells or control cells were used as target cells (E : T  : 1). All values were normalized relative to the control (radotinib 0 μM). The relative level was set to 1 for the control. (e) To further confirm the involvement of Fas-Fas ligand interaction in the radotinib-enhanced NK cytotoxicity, recombinant human soluble Fas was used to block Fas ligand on NK cells. Various concentrations of soluble Fas were preincubated with resting NK cells or radotinib-treated NK cells for 1 h, and then cytotoxicity assays were performed (E : T  : 1). All values were normalized relative to the control (radotinib 0 μM). The relative level was set to 1 for the control. Data are reported as . All values were analyzed by unpaired Student’s -tests using GraphPad Prism 5. , , and . All data presented are representative of three independent experiments.

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