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

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4.2. Changes in Caspase-3 Enzyme Activity and Loss of Mithocondrial Membrane Potantial) MMP To evaluate the apoptotic effects of Bortezomib on U-266 cells, Caspase-3 enzyme activity and mitochondrial membrane potential were two important apoptotic indicators. Treatment of U-266 cells for 72 h with 1, 10 and 20 nM Bortezomib resulted in 1.06, 1.13 and 1.17-fold increases, respectively, in caspase-3 activity as compared to untreated controls (Figure 4.2). Changes in Caspase-3 Enzyme Aktivity 200 150 100 50 0 100 Caspase-3 Enzyme Aktivity 106 113 117 C 1 10 20 Bortezomib (nM,72h) Figure 4.2. Changes in Caspase-3 Enzyme Activity We also assessed the loss of mitochondrial membrane potential in order to confirm the caspase-3 enzyme activity results and examine the roles of mitochondria in Bortezomib-induced apoptosis. Treatment of U-266 cells for 72 h with 1, 10 and 20 nM Bortezomib caused a significant loss of MMP (about 1.34, 1.85 and 2.14-fold, respectively, when compared with untreated controls), as measured by increased accumulation of cytoplasmic mitochondrial JC-1 (Figure 4.3). U-266 69
% Changes in Cytoplasmic/Mitochondrial JC-1 Figure 4.3. Changes in Cytoplasmic/Mitochondrial JC-1 in U-266 Cells Treated with Bortezomib 4.3. Two-Dimensional Polyacrylamide Gel Electrophoresis Results After determining the cytotoxic and apoptotic effects of Bortezomib on MM U-266 cells, total protein isolation was performed from Bortezomib and control group separately. After that isolated proteins were analyzed by Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Two-Dimensional Gel Electrophoresis, respectively to identify changes in protein profiles. Protein isolation and 2D PAGE experiments were repeated four times. Firstly, gels obtained from Bortezomib and control group were stained with CBB G-250, but the results were not satisfactory. Then CBB R-250 and R-350 was tried with another gels and with different staining procedures. Finally, a mass spectrometry compatible silver staining procedure was applied as explained in previous chapter to increase the sensitivity, because there was not enough protein that was visualized on the gels after Coomassie Brillant Blue Staining. The gel maps belonging to two different groups (Bortezomib (treated) and control (untreated)) indicated a broad distribution of spots in a pI range from 3.0 to 10.0 (Figure 4.4). 250 200 150 100 50 0 Mitochondrial Membrane Potential 100 134 185 214 C 1 10 20 Bortezomib (nM, 72h) U-266 70
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CHANGES IN PROTEIN PROFILES IN BORT
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ACKNOWLEDGEMENTS First of all, with
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ÖZET BORTEZOMĠB UYGULANAN MULTĠP
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2.1.4.1.2. Two-Dimensional Gel Elec
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LIST OF FIGURES Figure Page Figure
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LIST OF TABLES Table Page Table 1.1
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When normal cells in the body begin
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Figure 1.2. Development of Cancer S
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the myeloma cells does not help fig
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A rare form of MM called Nonsecreto
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1.2.3. Risk Factors for MM A risk f
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of the mineralizations by adjusting
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To measure the levels of red cells,
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The levels of blood urea nitrogen (
Page 29 and 30: By using the system introduced by D
Page 31 and 32: 1.2.7.2. Biology of The Proteasome
Page 33 and 34: Figure 1.10. Ubiquitin-Proteasome P
Page 35 and 36: Figure 1.12. Effect of Bortezomib o
Page 37 and 38: encompasses the investigation of pr
Page 39 and 40: of modification. Ubiquitin is a sma
Page 41 and 42: the given cells. On the other hand,
Page 43 and 44: is-acrylamide (N,N'-methylene-bisac
Page 45 and 46: 2.1.4.2. Detection of Protein Spots
Page 47 and 48: ange of 600-2500 Da. This is the id
Page 49 and 50: ions resolved by the mass analyzer
Page 51 and 52: 2.1.4.5.2. Mass Analyzers Figure 2.
Page 53 and 54: There is no doubt that different ma
Page 55 and 56: mask low-quantity proteins that may
Page 57 and 58: 3.1. Materials 3.1.1. Medias CHAPTE
Page 59 and 60: 3.2.5. Cell Proliferation Assay Ant
Page 61 and 62: After 72 hour incubation period at
Page 63 and 64: eceptors of the Tumor Necrosis Fact
Page 65 and 66: The preparation of stock BSA Standa
Page 67 and 68: 3.2.6.3. Detection of the Loss of M
Page 69 and 70: centrifugation, the supernatant was
Page 71 and 72: selected symmetrically to load the
Page 73 and 74: approximately 35 ml, the volume of
Page 75 and 76: After electrophoresis was finished,
Page 77 and 78: 7 Day Two: Reduction, Alkylation, a
Page 79: CHAPTER 4 RESULTS AND DISCUSSION 4.
Page 83 and 84: Table 4.1. Magnified Segments of Di
Page 85 and 86: Table 4.1. Magnified Segments of Di
Page 87 and 88: After labeling, black and red color
Page 89 and 90: 78 7 8 Y2S Y3S Table 4.2. Results o
Page 91 and 92: 80 12 S4Ca Table 4.2. Results of Di
Page 93 and 94: 82 27 Y5S Table 4.2. Results of Dif
Page 95 and 96: Spot 4, Ubiquitin-conjugating enzym
Page 97 and 98: is a transcriptional repressor, but
Page 99 and 100: activity and loss of mitochondrial
Page 101 and 102: distributed, but correlate with nat
Page 103 and 104: DeMartino, G. N. and Slaughter, C.
Page 105 and 106: proteomic analyses of a systematica
Page 107 and 108: Mahindraa, A., Hideshimab, T. and A
Page 109 and 110: Richardson, P. G., Hideshima, T. an
Page 111 and 112: Terpos, E. 2008. Bortezomib Directl
Page 113 and 114: A.1. RPMI-1640 Growth Medium APPEND
Page 115 and 116: Table B.1. Chemicals and Reagents U
Page 117 and 118: B.4.3. Standard Curve for BSA Table
Page 119 and 120: Urea (8M) 4.8 g CHAPS (%2) 0.2 g DT
Page 121 and 122: E. F. G. STOCK REAGENT PREPARATION
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