Figure_4.tif (4.02 MB)

Effect of Fe-SP or Cu-SP on viability, ROS generation, and induction of apoptic markers and MAPK expression.

figure

posted on 2013-02-20, 18:45 authored by Kyu Kwang Kim, Rakesh K. Singh, Robert M. Strongin, Richard G. Moore, Laurent Brard, Thilo S. Lange

(A) Cytotoxicity of Fe-SP or Cu-SP in NB cells. The viability assay was carried out after 24 h treatment of SMS-KCNR NB cells with 0–3 µM of Fe-SP, Cu-SP or respective metal salts. Experiments were performed in triplicates; data are expressed as the mean of the triplicate determinations (X±SD) of a representative experiment in % cell viability of untreated cells [ = 100%]. (B) Generation of intracellular Reactive Oxygen Species (ROS) after Fe-SP treatment. Generation of intracellular ROS following SMS-KCNR NB cells (large panels) or SKOV-3 OC control cells (small panels) after treatment for 4 h with 1.6 µM of Fe-SP or Cu-SP was measured by flow cytometry (see Materials and Methods). Data are presented as relative fluorescence intensity in a 2-dimensional FACS profile. Standardized gating was used for all samples. (C) Fe-SP cytotoxicity is blocked by antioxidant ascorbic acid. Cells were treated with with1.6 µM Fe-SP alone or in combination with antioxidant ascorbic acid. Viability of SMS-KCNR NB cells is presented as bar diagram and in percentages and of SKOV-3 OC control cells in percentages (insert). (D) Inhibiton of ROS generation in NB cells after Fe-SP treatment. Generation of intracellular ROS in SMS-KCNR NB was measured after treatment with 1.6 µM Fe-SP alone or in combination with antioxidant ascorbic acid (200 µM). (E) Expression of apoptotic markers and MAPK in NB cells after Fe-SP treatment with and without inhibiton of ROS generation. SMS-KCNR NB cells were treated with 0.8 µM Fe-SP in the absence (3, 6, 14, 24 h treatment, left panel) or presence of ascorbic acid (200 µM, 24 h treatment, right panel). Immunoblotting was carried out with primary antibodies against PARP-1, caspase-3, -7, pro-survival marker XIAP (inhibitor of effector caspases), and pro-apoptotic MAPK SAP/JNK or p38 in the active (phosphorylated) or inactive form. As an internal standard for equal loading blots were probed with an anti-GAPDH.