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. 2016 May 9;7(24):37064–37080. doi: 10.18632/oncotarget.9245

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Copyright: © 2016 Denoyer et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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A. TRAMP adenocarcinoma cells (TRAMP-C1) have normal intracellular copper levels. Total intracellular copper was measured in both TRAMP-C1 and mouse primary prostate epithelial cells (PrECs) cultured under basal conditions. Results are shown as copper (ng) per 10⁶ cells. B. TRAMP adenocarcinoma cells (TRAMP-C1) have elevated intracellular ROS levels. Intracellular ROS was measured using the cell permeable fluorogenic probe H₂DCF-DA and flow cytometry. Results represent mean fluorescence intensity (MFI) (geometric mean). C. Copper-ionophores potently kill TRAMP adenocarcinoma cells (TRAMP-C1). TRAMP-C1 cells were treated for 18 hours with Cu^II(gtsm), disulfiram (DSF) or clioquinol alone or in combination with 20 μM CuCl₂. Ionophore concentrations are shown and cell viability was determined by the propidium iodide exclusion assay and flow cytometry. D. Copper-ionophores selectively kill TRAMP adenocarcinoma cells while not affecting the viability of mouse primary prostate epithelial cells (PrECs). Both cell lines were treated for 18 hours with Cu^II(gtsm), disulfiram or clioquinol in combination with 20 μM CuCl₂. Ionophore concentrations are shown and cell viability was determined by the propidium iodide exclusion assay and flow cytometry. E. Copper-ionophores generate intracellular ROS in TRAMP adenocarcinoma cells (TRAMP-C1) (i), but not in mouse primary prostate epithelial cells (PrECs) (ii). Both cell lines were treated for 2, 4, 6 or 18 hours with Cu^II(gtsm), disulfiram or clioquinol in combination with 20 μM CuCl₂. Ionophore concentrations are shown and intracellular ROS was measured using the cell permeable fluorogenic probe H₂DCF-DA and flow cytometry. Results represent mean fluorescence intensity (MFI) (geometric mean) F. TRAMP adenocarcinoma cells (TRAMP-C1) have markedly reduced antioxidant capacity. Reduced (GSH) (i) and oxidised (GSSG) (ii) glutathione were measured in TRAMP adenocarcinoma cells (TRAMP-C1) and mouse primary prostate epithelial cells (PrECs) by HPLC. (iii) The GSH:GSSG ratio is compared between both cell lines. G. Differential GSH expression in TRAMP adenocarcinoma cells (TRAMP-C1) treated with copper-ionophores. Reduced glutathione (GSH) was measured in mouse primary prostate epithelial cells (PrECs) (i) and TRAMP adenocarcinoma cells (TRAMP-C1) (ii) following treatment for 18 hours with sublethal concentrations of Cu^II(gtsm) (20 nM), disulfiram (150 nM) or clioquinol (2 μM) (with 20 μM CuCl₂). Glutathione (GSH & GSSG) was measured by HPLC. Results represent mean ± STDEV (bar) of triplicate determinations for each measurement. (*p < 0.05; **p < 0.01).