Figure 3

Inhibition of proteasome activity and apoptosis induction in MDA-MB-231 breast cancer cells by clioquinol (CQ) + copper and pyrrolidine dithiocarbamate (PDTC) + copper. MDA-MB-231 breast cancer cells were treated with 20 μM copper (Cu), CQ, CQ + copper (CC), tetrathiomolybdate (TM), TM + copper (TC), or 10 μM PDTC (P), or PDTC + copper (PC), using DMSO (DM) as a control. Cells were collected after 24 h treatment and analyzed for proteasome inhibition. (a) Proteasome activity as measured in released fluorescence units (RFUs) by release of 7-amino-4-methylcoumarins (AMCs) from substrate specific for chymotrypsin-like activity. (b) Western analysis for accumulation of ubiquitinated proteins as an indicator of proteasome inhibition. Treatment with PDTC + copper (PC; 10 μM) or CQ + copper (CC; 20 μM) results in reduced release of AMCs and ubiquitinated protein accumulation, suggesting proteasome inhibition. (c) Western analysis for cleavage of poly(ADP ribose) polymerase (PARP) as an indication of apoptosis. Treatment with CQ + copper (20 μM) or PDTC + copper (10 μM) results in cleavage of PARP, indicating that these complexes are capable of inducing apoptosis.