The influence of disulfiram complex with copper (CuEt) on cellular proteasome in
breast cancer cell lines
Skrott Z1*, Dou QP2, Cvek B1, 1Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Olomouc, Czech
Republic; 2Barbara Ann Karmanos Cancer Institute, Departments of Oncology, Pharmacology and Pathology, School of Medicine, Wayne
State University, Detroit, MI, USA; zdenek.skrott@centrum.cz
CT-like activity [%]
Introduction: Disulfiram (Antabuse), a drug commonly used for alcohol aversion therapy, shows promising anticancer activity. Disulfiram´s effectiveness
as a cancer therapeutics is attributed to its copper complex formed in human body in the presence of copper ions. Disulfiram-copper complex is a
member of metal-based inhibitors of the proteasome (Chen et al. Cancer Res, 2006), a multisubunit complex responsible for degradation of cellular
proteins. Proteasome provides a suitable anticancer target with first-in-class drug bortezomib (Velcade). Unexpectedly, synthetic disulfiram-copper
complex (CuEt) has little inhibitory effect on the purified 20S proteasome (Cvek et al. J Med Chem, 2008) in comparison to common proteasome
inhibitors like bortezomib. We hypothesize that the target of CuEt is the 19S regulatory particle of the 26S proteasome, such as cryptic deubiquitinase
Poh1 located in the lid of the 19S proteasome. This hypothesis will be investigated in the near future. Here, we measure the inhibition of the 20S
proteasome in breast cancer cell lines MCF7 and MDA MB 231 after treatment with CuEt or bortezomib. We have also compared the effect of
bortezomib and CuEt on some phenomena related to proteasome inhibtion, such as accumulation of ubiquitinated conjugates or accumulation of
common proteasome substrate, I-κBα.
Results: We have previously reported that synthetic complex CuEt does not inhibit CT-like
120
activity
of
the
purified
20S
proteasome.
To
examine
the
influence
of
CuEt
and
bortezomib
on
A 100
the 20S proteasome in cells, we incubated MCF7 and MDA MB 231 cell lines with CuEt or
bortezomib. After treatment, lyzates were prepared for measurement of proteasomal CT-like
80
activity
and
Western
blot
analysis.
Compare
to
bortezomib,
CuEt
complex
had
no
or
little
MCF7 CuEt
60
MCF7 Bort
inhibitory effect on CT-like activity of the 20S proteasome in time-dependent (Fig. 1A) and
MDA MB 231 CuEt
40
dose-dependent (Fig. 1B) manner in both cell lines. Inhibition of cellular proteasome is
MDA MB 231 Bort
associated
with
accumulation
of
ubiquitinated
proteins.
To
determine
whether
CuEt
inhibits
20
whole 26S proteasome, we detected level of ubiquitinated conjugates. We observed time0
dependent (Fig. 2A) and dose-dependent (Fig. 2B) accumulation of ubiquitinated proteins in
1
3
6
9
time [hr]
both cell lines. Next, we examined the effect of CuEt and bortozemib on TNFα-induced
degradation of I-κBα. Both, CuEt and bortezomib caused accumulation of I-κBα in
120
comparison to control in MCF7 and MDA MB 231 cell lines (Fig. 3).
B
A
CT-like activity [%]
100
B
80
MCF7 CuEt
MCF7 Bort
MDA MB 231 CuEt
MDA MB 231 Bort
60
40
20
0
0.1
0.5
1
5
concentration [uM]
Fig. (1). Time-dependent (A) and dose-dependent (B) inhibition of the 20S proteasome. MCF7
and MDA MB 231 cells were incubated with 1 μM CuEt or bortezomib for indicated time (A) or
with various concentrations of CuEt or bortezomib for 3 hours (B). DMSO was used as control.
Fig. (3). Accumulation of
I-κBα . MCF7 and MDA
MB 231 cells were
treated with CuEt or
bortezomib at indicated
concentrations for 3
hours. After that, cells
were incubated for 15
min with 10 ng/ml TNFα,
followed by preparation
of whole-cell lyzates for
proteasomal and WB
assays.
Methods: Inhibition of 20S proteasome in cell extracts. Whole-cell lyzates (10 μg) from cells
treated as indicated were incubated for 2 hours in 100 μl assay buffer (50 mM Tris-HCl, pH 7.5)
with 20 μM fluorogenic substrate Suc-LLVY-AMC for proteasomal chymotrypsin-like activity.
Western blot analysis. Whole-cell lyzates (40 μg) from cells treated as indicated were
separated by SDS-PAGE and transferred to nitrocelulose membrane. Proteins were
immunoblotted with specific antibodies against ubiquitin, β-actin, I-κBα and visualized by
enhanced chemiluminescence.
Fig. (2). Time-dependent (A) and dose-dependent (B) accumulation of ubiquitinated proteins. MCF7 and MDA MB 231 cells were incubated with 1 μM
CuEt for indicated time (A) or with various concentrations of CuEt for 3 hours (B). DMSO was used as control.
Discussion: Disulfiram-copper complex (CuEt) is member of metal-based proteasome
inhibitors. Based on our previous observations, we have hypothesized that target of CuEt is
different from bortezomib, possibly the 19S regulatory particle of the 26S proteasome. In
this study, we showed that under the tested conditions, CuEt does not significantly inhibit
CT-like activity of the 20S proteasome in breast cancer cell lines. On the other hand, after
treatment with CuEt we observed hallmarks of proteasomal inhibition, such as accumulation
of ubiquitinated proteins or accumulation of well described proteasome substrate, I-κBα.
Taken together, these results indicate that one of the targets of CuEt is the 19S proteasome.
Conclusion: Finally, data presented here support our hypothesis that synthetic disulfiramcopper complex (CuEt) inhibits the proteasome by a mechanism that is distinct to
bortezomib.
Acknowledgement: This work was financed by project OP VK CZ.1.07/2.3.00/20.0062 “An inexpensive drug Antabuse as anticancer remedy: mechanism of action and clinical trials” from
resources of European Union and the Czech Republic.