Induction of apoptosis in premalignant-MCF10 breast cells and proteasome inhibition and apoptosis induction in breast cancer MDA-MB-231 cells cultured to contain elevated copper and post-treated with clioquinol (CQ) and pyrrolidine dithiocarbamate (PDTC). (a,c) Premalignant-MCF10 (KCL2) cells were cultured in media containing 25 μM copper for two weeks. (b,d,e) MDA-MB-231 cells were cultured in 25 μM copper for 48 h. After culturing, cells were washed with PBS and treated with media containing: (a) 1, 10, or 50 μM CQ; (b-e) 25 μM CQ; 25 μM tetrathiomolybdate (TM); or (b,d,e) 10 μM PDTC. An equivalent volume of DMSO (DM) was used as control. KCL2 and MDA-MB-231 cells were examined for (a,b) apoptotic morphology and (c,e) PARP cleavage. (d) MDA-MB-231 cells were also examined by western blot for accumulation of ubiquitinated proteins. (a,c) KCL2 cells containing clinically relevant levels of copper were sensitive to treatment by CQ alone, which induced apoptosis. (c) KCL2 cells cultured under standard conditions were resistant to treatment by 25 μM CQ. Similarly, MDA-MB-231 cells cultured to contain elevated copper were sensitive to CQ or PDTC and underwent proteasome inhibition as measured by accumulation of (d) ubiquitinated (Ub) proteins and apoptosis as evidenced by morphology and (b,e) poly (ADP ribose) polymerase (PARP) cleavage. These data suggest that KCL2 and MDA-MB-231 cells cultured to contain clinically relevant levels of copper are sensitive to treatment with CQ or PDTC alone, but not TM.