Figure 6

Therapeutic doses of CuPT inhibits the proteasome function in cultured cells.

(a and b) CuPT induced the accumulation of ubiquitinated proteins. MCF-7 and U266 cells were treated either with CuPT (0.5, 0.75, 1.0 μM) for 12 hours or CuPT (0.5 μM) for various times (12, 18, 24 hours) and then ubiquitinated proteins were detected by Western blot. Dose- or time-dependent effect of CuPT was shown in (a) and (b), respectively. (c) CuPT induced the accumulation of K48-linked poly-ubiquitinated proteins. HepG2 cells were exposed to CuPT as indicated for 12 h, ubiquitinated and K48-linked proteins were detected by Western blot. (d) CuPT induced the accumulation of GFPu, a surrogate proteasome substrate. HEK-293 cells harboring GFPu were treated with CuPT for 12 hours and then ubiquitinated proteins and GFP protein were detected by Western blot or imaged under an inverted fluorescence microscope. Bortezomib/Velcade (Vel) was used a positive control. (e) CuPT induced the accumulation of ubiquitinated proteins in primary cultured cancer cells. Cancer cells from AML patients or normal controls were cultured and treated with various doses of CuPT and Vel for 6 hours and then the ubiquitinated proteins were detected by Western blot. (f) The effect of CuPT on proteasome peptidase activity in situ. HepG2 cells or U266 cells were treated with increasing doses of CuPT and Vel (10 nM) for 6 hours, followed by addition of proteasome substrates to the treated cells and then peptidase activities including CT-like, caspase-like and trypsin-like were detected in situ. Mean ± SD (n = 3). *P < 0.05, versus control-treated. (g) The effect of CuPT on proteasome peptidase activities in vitro. 20S proteasome was treated with increasing doses of CuPT or Vel in vitro and then proteasome peptidase activities were detected.