changes in protein profiles in bortezomib applied multiple myeloma ...
changes in protein profiles in bortezomib applied multiple myeloma ...
changes in protein profiles in bortezomib applied multiple myeloma ...
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eceptors of the Tumor Necrosis Factor (TNF) results <strong>in</strong> rapid activation of the <strong>in</strong>itiator<br />
caspase-8 mak<strong>in</strong>g a scaffold after its recruitment to a trimerized receptor-ligand<br />
complex (DISC) through the adaptor molecule Fas-Associated Death Doma<strong>in</strong> Prote<strong>in</strong><br />
(FADD) (Sartorius et al., 2001). Then activated caspase-8 cleaves and thereby activates<br />
caspase-3 for the execution of apoptosis (Gewies, 2003).<br />
Changes <strong>in</strong> caspase-3 enzyme activity of MM U-266 cells <strong>in</strong> response to<br />
Bortezomib were evaluated by us<strong>in</strong>g caspase-3 colorimetric assay kit (R&D Systems,<br />
USA) as described previously (Baran et al., 2007). This assay is based on<br />
spectrophotometric detection of the chromophor p-nitroanilide (pNA) after cleavage<br />
from the labeled substrate DEVD- pNA . The pNA light emission can be quantified at<br />
405 nm.<br />
In short, 1x10 6 cells were seeded <strong>in</strong> a 6-well plate <strong>in</strong> 2 ml growth medium <strong>in</strong> the<br />
absence or presence of <strong>in</strong>creas<strong>in</strong>g concentrations of Bortezomib (1 nM, 10 nM, 20 nM)<br />
at 37°C <strong>in</strong> 5% CO2 for 72 hours. Untreated cells were used as control group.<br />
Figure 3.3. Aplied Bortezomib Doses on U-266 Cells for Caspase-3 Enzyme Activity<br />
In Figure 3.3 ;<br />
C<br />
20 nM<br />
1 nM<br />
10 nM<br />
C is correspond to control group (1x10 6 U-266 Cells / 2 ml / well),<br />
1 nM is correspond to 1 nM Bortezomib concentration (1x10 6 U-266<br />
Cells / 2 ml / well + 1 nM Bortezomib <strong>in</strong> 2 ml),<br />
10 nM is correspond to 10 nM Bortezomib concentration (1x10 6 U-266<br />
Cells / 2 ml / well + 10 nM Bortezomib <strong>in</strong> 2 ml),<br />
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