Transcript Snímek 1

Differential activity of synthetic Au (III) and Pt (II) diethyldithiocarbamate
complexes towards the proteasome in hepatoma cell line SK-Hep1
Jindrich Sedlacek1, Jan Taraba2, Boris Cvek1
1 Department of Cell Biology & Genetics, Palacky University, Slechtitelu 11, Olomouc 78371;
2 Department of Chemistry, Episcopal High School, Barvicova 85, Brno 60200, Czech Republic
INTRODUCTION
Metal compounds have been used for treatment diseases for centuries, although mechanism of their biological action were unknown. More than half a century
ago, it was observed that platinum compounds were binding to DNA, thereby altering their function. This is a beginning new promising of cancer treatment
using metal-based drugs. After successes achieved with platinum-based drugs, such as cis-platin, against many types of cancers. Using of platinum-based
drugs were associated with serious clinical problems, such as tissue toxicity and drug resistance. This is the main reason to search new metal-compunds that
might produce more specific antitumor efects. Nowadays, a new gold and platinum drug constitute promising agents for cancer treatment. Some of these
compounds were shown activity against proteasome in various human tumor cell lines. We synthesized diethyldithiocarbamate complexes with platinum (II)
and gold (III) and described them by X-ray diffraction and tested in hepato-carcinoma derived cell line SK-Hep1.
RESULTS
We have shown that only The gold (III) diethyldithiocarbamate complex anb bortezomib target the tumor cellular proteasome (Fig.-2 and Fig.-3). These
properties of test compounds caused growth inhibition efect (Tab.-1). Inhibition of protesome by gold complex is associated with accumulation of ubiquitinated
proteins (Fig.-5 ). Gold complex and bortezomib caused accumulation of proteasome dependent proteins IκBα, p27, p53. PARP cleavage (Fig.- 6) indicates that
proteasome inhibition is associated with apoptosis of the cells. In contrast, platinum (II) diethyldithiocarbamate complex is inactive in the tested parameters.
confirmed by using X-ray single-crystal diffraction
Tab.-1. IC50 value of [AuIII(DEDTC)3], [PtII(DEDTC)2] and bortezomib were determined
in SK-Hep1 cells after 24 hours of treatment
IC50 (μM)
IC50 (μM)
[AuIII(DEDTC) 3]
5,23
6,07
5,62
[PtII(DEDTC) 2]
bortezomib
>15
>15
>15
0,627
1,187
0,388
mean
CT-like activity (%)
100
50
90
60
30
0
0
PtEt 10 μM
AuEt 10 μM
AuEt 5 μM
Bortezomib 1 μM
Fig.-2. Influence of 20S proteasome CT-like activity by
[AuIII(DEDTC)3], [PtII(DEDTC)2], and bortezomib in
cells lysat . Lysats (10 g) were preincubated with
compounds for 15 minutes, followed by an additional
2-h incubation with the fluorogenic substrate
Fig.-1. Molecular structures of both [AuIII(DEDTC)3] and [PtII(DEDTC)2]
IC50 (μM)
120
150
CT-like activity (%)
METHODS
Growth inhibition assay. Cells were cultured for 24 hours with synthetic coplexes. In
parallel, the cells were treated with (DMSO; 0.001%) and Triton X-100 (5%) to assess the
minimal and maximal cell damage, respectively. Cell viability was determined by MTT
method using the standart protocol. Absorbance was measured spectrophotometrically
at 570 nm. The values of IC50 were calculated using data from three independent cell
passages.
Inhibition of 20S proteasome in cell extracts. Whole-cell lyzates (10 μg) from cells
treated as indicated were incubated for 120 minutes in 90 μl assay buffer with 20 μM
substrate Suc-LLVY-AMC for detektion CT-like activity of proteasom.
Immunoblot analysis. Whole-cell lyzates (50 μg) from cells were separated by SDS-PAGE
and transferred to 0,45 0,45 μM PVDF membrane. Proteins were detekt by specific
antibodies against ubiquitin, PARP, p53, p27 and IκBα. β-actin was used as loading
control.
PtEt 10 μM
AuEt 5 μM
AuEt 10 μM
Fig.-3. Inhibition of proteasomal CT-like activity in human
SK-Hep1 cancer cells for 24 hours. Follows, cell extracts
were prepared by lysis in buffer. Extracts (10 μg) were
incubated with the fluorogenic substrate
Fig.-6. PARP fragment (89 kDa). SK-hep1 cells
were treated with complexes, bortezomib and
solvent (DMSO) for 8 hours
SD
5,64
-
0,42
-
0,734
0,41
Fig.-5. Inhibition of degradation some
important cellular proteins IκB-α, p27,
p53. Cells were treated with
bortezomib, complexes and solvent
(DMSO) for 8 hours
Fig.-4. Accumulation of ubiquitinate
proteins. SK- Hep1 cells were treated
with solvent (DMSO), bortezomib
and complexes for ½ hours
CONCLUSION
The gold (III) diethyldithiocarbamte complex, but not the platinum (II) complex, inhibited chymotrypsin-like activity of purified and celluar 20S proteasome.
The antitumor activity of gold (III) complexes is associated with proteasomal activity inhibition, with the accumulation of proteasome target proteins, p27,
IκBα and stabilizes the tumor suppressor protein p53. Cytotoxicity of gold complex is associated with apoptosis apoptosis as showed PARP cleavage. The
cell effect of these group of compounds is still partly unknown, although screening and development of organometallic compounds as anticancer drugs can
provide promising results. The gold(III) dithiocarbamate complex act as proteasome inhibitors and has a potential to become antitumor drug.
ACKNOWLEDGMENT
Our laboratory is supported by grant 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