changes in protein profiles in bortezomib applied multiple myeloma ...

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