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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After 72 hour <strong>in</strong>cubation period at 37°C <strong>in</strong> 5% CO2, they were treated with 50 µl<br />
XTT reagent for 4 hours at CO2 <strong>in</strong>cubator. After that, the absorbances of the samples<br />
were read under 492 nm wavelength of light by Elisa reader (Thermo Electron<br />
Corporation Multiskan Spectrum, F<strong>in</strong>land) and graphed cell proliferation plots. F<strong>in</strong>ally,<br />
IC50 value (the concentration of drug that <strong>in</strong>hibits 50% of cell proliferation as<br />
compared to untreated control) of Bortezomib was calculated from cell proliferation<br />
plots.<br />
3.2.6. Evaluation of Apoptosis<br />
3.2.6.1. Measurements of Caspase-3 Enzyme Activity<br />
Also called programmed cell death or cell suicide, apoptosis is an <strong>in</strong>tegral and<br />
necessary part of life cycle of organisms. In the human body, about a hundred thousand<br />
cells are produced every second by mitosis and a similar number of them die by<br />
apoptosis (Vaux and Korsmeyer, 1999). The apoptotic mode of cell death can be<br />
described as a process which plays an important role <strong>in</strong> the improvement and<br />
homeostasis of multicellular organism and <strong>in</strong> the regulation and ma<strong>in</strong>tenance of the cell<br />
populations <strong>in</strong> tissues upon pathological and physiological conditions (Jacobson et al.,<br />
1997; Meier et al., 2000; Hengartner, 2000; Leist and Jaattela, 2001). Apoptosis can be<br />
triggered by various stimuli from outside or <strong>in</strong>side the cell. For example, ligation of cell<br />
surface receptors, DNA damage as a cause of defects <strong>in</strong> DNA repair mechanisms,<br />
treatment with cytotoxic drugs or irradiation, a lack of survival signals, contradictory<br />
cell cycle signal<strong>in</strong>g or developmental death signals (Gewies, 2003). It is doubtless that<br />
apoptotic processes are of widespread biological and biochemical significance. A major<br />
biochemical feature of apoptosis is the sequential activation of the caspases (cyste<strong>in</strong>e-<br />
dependent aspartate- specific proteases), a family of proteases whose substrate <strong>in</strong>clude<br />
large prote<strong>in</strong> precursors of enzymes capable of destroy<strong>in</strong>g DNA (endonucleases); lam<strong>in</strong><br />
and act<strong>in</strong> (proteases); and prote<strong>in</strong>s <strong>in</strong>volved <strong>in</strong> DNA repair<strong>in</strong>g, RNA splic<strong>in</strong>g, signal<br />
transduction and transcription factors (Thornberry and Lazebnik, 1998; Hengartner,<br />
2000; Green 2000; K<strong>in</strong>g and Rob<strong>in</strong>s, 2006).<br />
One of the vital members of these caspases family is Caspase-3 enzyme which is<br />
the key component <strong>in</strong> that it takes part <strong>in</strong> the center of apoptotic pathways (Bratton et<br />
50