the anticancer mechanism of ibuprofen or indomethacin in colorectal
TRANSCRIPT
THE ANTICANCER MECHANISM OF IBUPROFEN OR INDOMETHACIN IN COLORECTAL CANCER
CELLS
KHOO BOON YIN
UNIVERSITI SAINS MALAYSIA 2007
Acknowledgements
I would like to extend my thanks and gratitude to my supervisor,
Associate Professor Dr. Soriani Yaacob and co-supervisor, Professor Norazmi
Mohd. Nor for their guidance and support in this project. I am grateful to be
given the opportunity to carry out my research project under their supervision. I
am also grateful for the help and advice given by all the academic and non-
academic staffs of the Department of Microbiology, PPSK, INFORMM and
everyone else who had lend me a hand in my project. It has been a wonderful
experience working with all of you. My sincere thanks to MOSTE for the
National Science Fellowship. Special thanks to Prof. Smith M.-L. (University of
Leeds, UK), Prof. Suzanne Cory (Walter & Eliza Hall Institute of Medical
Research, Australia) and Prof. Shigeaki Kato (University of Tokyo, Japan) in
providing the clones for this study. To my parents and friends, I thank them for
their sound advice and relentless support.
Abbreviations
Aberrant crypt foli ACF Adenomatous polyposis coli APC Adenosine 5’-triphosphate ATP American Type Culture Collection ATCC Ammonium persulfate APS Apoptosis inducing factor AIF Apoptosis protease activating factor-1 Apaf-1 Arachidonic acid AA Azoxymethane AOM Base pair bp B-cell lymphoma 2 Bcl-2 Bcl-2 associated X protein Bax Bcl-2-associated death promoter homologue Bad Beta-mercaptoethanol β-ME Bovine serum albumin BSA Caspase recruitment domain CARD Chinese hamster ovary cells CHO c-jun N-terminal kinase JNK Colorectal cancer CRC COX-2-negative mice COX-2-/- mice COX-2-positive mice COX-2+/+ miceCyclooxygenase COX Cytokeratin-18 CK-18 Cytotoxic T-lymphocytes CTLs Death domains DD Death effector domain DED Death inducing signaling complex DISC Death receptor 4 and death receptor 5 DR4 and DR5 Diethylpyrocarbonate DEPC Dimethyl sulfoxide DMSO Docosahexanoic acid DHA Dulbecco’s Modified Eagle’s Medium DMEM Effective concentration that causes 50% of drug response EC50Ethidium bromide EtBr Ethylenediamine-tetra acetic acid EDTA Fas-associated death domain FADD Fetal bovine serum FBS European Collection of Cell Cultures ECACC Glycosylphosphatidylinositol GPI Hour h Horseradish peroxidase HRP in situ end labeling ISEL Inhibitors of apoptosis proteins IAPs Insulin-like growth factors I and II IGF I and II Isopropyl-beta-D-thiogalactopyranoside IPTG Kilo Dalton kDa Lactate dehydrogenase LDH
Ligand binding domain LBD Molecular weight MW N,N,N’N’-Tetra-methylethylenediamine TEMED Necrosis factor-kappa B NF-κB Non-steroidal anti-inflammatory drugs NSAIDs Optical density OD Peroxisome proliferator-activated receptors PPARs Peroxisome proliferators-activated receptor response elements
PPRE
Peroxisome proliferators-activated receptor-alpha PPARα Peroxisome proliferators-activated receptor-delta PPARδ Peroxisome proliferators-activated receptor-gamma PPARγ Phenylbutyrate PB Phenylmethylsulfonyl flouride PMSF Phosphate-buffered saline PBS Phosphoinositol -3-kinase PI3K Poly ADP-ribose polymerase PARP Prostaglandins PGs Receptor-interacting protein RIP Retinoid X receptor RXR RIP-associated ICH-1/CED-3 homologous protein with a DD RAIDD Roselle’s Park Memorial Institute Medium RPMI Serine/threonine kinase Akt Sodium citrate SOC Sodium dodecyl sulphate SDS Standard error of the mean SEM T-cell factor TCF Tdt-mediated dUTP nick end labeling TUNEL Thiazolidinediones TZD TNF receptor-1 TNF-R1 TNFα-related apoptosis-inducing ligand TRAIL TNFalpha TNFα TNF-associated factor 2 TRAF-2 TNFR-associated death domain TRADD Tris borate EDTA TBE Tumor necrosis factor TNF Tumor necrosis factor-alpha TNFα volume/volume v/v weight/volume w/v X-galactosidase X-gal
List of contents
PageAcknowledgement ii List of contents iii List of Tables vii List of Figures viii Abbreviations x Abstract xii Abstrak
xvi
Chapter 1 Introduction……………………………………………………..... 1 1.1 Introduction of NSAIDs……………………………………………………. 1.2 The non-selective COX inhibitors………………………………………... 1.3 Evaluation of NSAIDs as PPARγ ligands……………………………….. 1.4 The role of PPARγ ligands in cancer……………………………………..1.5 Apoptosis…………………………………………………………………… 1.6 Morphological features of necrosis and apoptosis…………………...... 1.7 The putative mechanisms of PPARγ ligand-induced apoptosis……….
2 5 6 9 11 12 14
1.7.1 Mitochondrial-dependent apoptosis……………………………. 1.7.1.1 Caspases activation in apoptosis…………………... 1.7.2 Death receptor-ligand apoptosis………………........................
15 19 24
1.7.2.1 Signaling by CD95/Fas……………………………...1.7.2.2 Signaling by TNF-R1………………………………...1.7.2.3 Induction of apoptosis by TRAIL…………………...
24 25 29
1.7.3 The survival signaling pathway….……………………………... 31 1.8 Ibuprofen and indomethacin……………………………………………… 1.9 The molecular mechanism of action of NSAIDs…….………………....
33 36
1.9.1 Mutation of APC gene in Wnt signaling pathway……………... 1.9.1.1 Down-regulation of β-catenin by NSAIDs……….……. 1.9.2 The effect of NSAIDs on PPARδ expression….……………....
39 40 41
1.10 Aims of the study…………………………………………………………
41
Chapter 2 Materials and Methods…………………………………………. 46 2.1 Materials……………………………………………………………………..
45
2.1.1 Chemicals and reagents……………………………………….... 2.1.2 Kits and consumables………………………………………….... 2.1.3 Primers and probes used for Real-time PCR…………………. 2.1.4 Antibody used for Western blotting analysis…………...……...
45 47 48 50
2.2 Cell culture…………………………………………………………….......... 53 2.2.1 Human CRC cell lines………………..…………………………..
2.2.2 Preparation of reagents for cell culture work………………….. 2.2.3 Culture procedures and conditions……………………………..
53 53 58
2.2.3.1 Thawing of cells from frozen storage………………2.2.3.2 Sub-culturing of cells……………………………….. 2.2.3.3 Viability test and counting the number of cells……2.2.3.4 Mycoplasma contamination test…………………… 2.2.3.5 Freezing down of cells……………………………....
58 59 60 61 61
2.3 Cytotoxicity determination…………………………………………………. 62 2.3.1 Determination of the optimal cell concentration……………….
2.3.2 Preparation of NSAID stock solutions…………………………. 2.3.3 Treatment of cells for LDH assay………………………………. 2.3.4 Preparation of LDH assay reaction mixture…………………....2.3.5 Determination of LDH activity…………………………………...
63 65 66 70 70
2.4 Apoptosis detection………………………………………………………... 72 2.4.1 Preparation of buffers and reagents for apoptosis detection...
2.4.2 Treatment of cells for apoptosis detection…………………….. 2.4.3 Detection of apoptosis…………………………………………… 2.4.4 Flow cytometry…………………………………………………....
72 73 73 74
2.5 Western blotting..............………………………………………………….. 76 2.5.1 Preparation of buffers and reagents for protein extraction…...
2.5.2 Treatment of cells for protein preparation……………………... 2.5.3 Extraction of protein……………………………………………… 2.5.4 Protein assay……………………………………………………... 2.5.5 Preparation of buffers and reagents for Western bloting……. 2.5.6 SDS-PAGE……………………………………………………….. 2.5.7 Transfer of proteins onto nitrocellulose membrane…………... 2.5.8 Antibodies and ECL detection.………………………………..
76 78 80 81 82 85 86 86
2.6 Agarose gel electrophoresis………………………………………………. 88 2.6.1 Buffers and reagents for agarose gel electrophoresis………..
2.6.2 Preparation of agarose gel……………………………………… 2.6.3 Agarose gel electrophoresis……………………………………..
88 90 90
2.7 Preparation of total cellular RNA and cDNA samples………………….. 91 2.7.1 Treatment of cells for the preparation of total cellular RNA.....
2.7.2 Extraction of total cellular RNA……………………………….....2.7.3 Measurement of RNA purity and concentration…………….....2.7.4 Synthesis of first-strand cDNA………………………………….. 2.7.5 Confirmation of cDNA synthesis………………………………...
91 91 92 93 93
2.8 Construction of endogenous standards for Real-time PCR………….... 94 2.8.1 Preparation of reagents for cloning……………………………..
2.8.2 Preparation of E. coli competent cells………………………..... 2.8.3 Transformation of competent cells……………………………... 2.8.4 Screening of positive clones by PCR………………………….. 2.8.5 Extraction of plasmid DNA……………………………………….2.8.6 Sequencing……………………………………………................ 2.8.7 Measurement of DNA purity and concentration…………….....2.8.8 Preparation of E. coli glycerol stock…………………………....
94 96 97 101 101 102 103 103
2.9 Real-time PCR…………………………………………………………….... 104 2.9.1 Reagents for Real-time PCR…………………………………….
2.9.2 Real-time PCR set-up………………………………………….... 104 105
2.10 Statistical analysis……………………………………………………….
106
Chapter 3 Cytotoxic effect of ibuprofen or indomethacin in human CRC cell lines………………………..........……………………..
107
3.1 Introduction…………………………………………………………………..3.2 Experimental design……………………………………………………….. 3.3 Results…………………………………………………………………….....
108 113 114
3.3.1 The COX-2 mRNA expression in CRC cell lines…………….... 3.3.2 The cytotoxic effect of ibuprofen or indomethacin in HCA-7
cells…………………………………………………………………..3.3.3 The cytotoxic effect of ibuprofen or indomethacin in HT-29
cells…………………………………………………………………..3.3.4 The cytotoxic effect of ibuprofen or indomethacin in HCT-116
cells……………………………………………………….......…….. 3.3.5 Determination of the constant EC50 values of ibuprofen or
indomethacin on HCA-7, HT-29 and HCT-116 cells……..…....
114 117 120 122 125
3.4 Discussion…………………………………………………………………...
128
Chapter 4 Cell death induced by ibuprofen or indomethacin in human CRC cell lines…………....……………………………...
131
4.1 Introduction………………………………………………………………… 4.2 Experimental design……………………………………………………… 4.3 Results……………………………………………………………..............
132 135 137
4.3.1 The percentage of apoptotic cells induced by ibuprofen or indomethacin in HCA-7 cells……….......................................
4.3.2 The percentage of apoptotic cells induced by ibuprofen or indomethacin in HT-29 cells…………………………………….
4.3.3 The percentage of apoptotic cells induced by ibuprofen or indomethacin in HCT-116 cells…….......................................
4.3.4 Optimisation of Western blot analysis…………...................... 4.3.5 The expression of pro-caspase-8, -9, -3 and -7 in HCA-7
cells following treatment with ibuprofen or indomethacin……………………………………........................
4.3.6 The expression of pro-caspase-8, -9, -3 and -7 in HT-29 cells following treatment with ibuprofen or indomethacin………………….................................................
4.3.7 The expression of pro-caspase-8, -9, -3 and -7 in HCT-116 cells following treatments with ibuprofen or indomethacin…………………….............................................
137 139 141 143 143 145 147
4.4 Discussion…………………………………………………………………..
149
Chapter 5 Modulation of COX-2, c-myc, β-catenin and TCF-4 mRNA expression by ibuprofen or indomethacin in human CRC cell lines...............................................................................…
154
5.1 Introduction…………………………………………………………………..5.2 Experimental design……………………………………………………….. 5.3 Results……………………………………………………………………….
155 157 158
5.3.1 Confirmation of the specificity of the PCR amplification…….. 5.3.2 Construction of standard curves for COX-2, c-myc, β-catenin
and TCF-4………………………………………………………... 5.3.3 Quality of total cellular RNA extraction……………………...... 5.3.4 Confirmation of cDNA samples………………. 5.3.5 Expression of COX-2, c-myc, β-catenin and TCF-4 mRNA in
HCA-7 cells following treatments with ibuprofen or indomethacin…………………………......................................
158 162 164 164 167
5.3.6 Expression of COX-2, c-myc, β-catenin and TCF-4 mRNA in HT-29 cells following treatment with ibuprofen or indomethacin…………………………......................................
5.3.7 Expression of COX-2, c-myc, β-catenin and TCF-4 mRNA in HCT-116 cells following treatment with ibuprofen or indomethacin……………………………………………………..
171 174
5.4 Discussion…………………………………………………………………..
177
Chapter 6 Modulation of PPARα, PPARδ, PPARγ1 and PPARγ2 mRNA expression by ibuprofen or indomethacin in human CRC cell lines………………………...............................……………..
183
6.1 Introduction…………………………………………………………………..6.2 Experimental Design………………………………………………………..6.3 Results…………………………………………………………………….....
184 186 188
6.3.1 Confirmation of the specificity of PCR amplification of PPARα, PPARδ, PPARγ1 and PPARγ2……………………….
6.3.2 Construction of standard curves of PPARα, PPARδ, PPARγ1 and PPARγ2…………………………………………...
6.3.3 Expression of PPARα, PPARδ, PPARγ1 and PPARγ2 mRNA in HCA-7 cells following treatments with ibuprofen or indomethacin…………………………………………………..
6.3.4 Expression of PPARα, PPARδ, PPARγ1 and PPARγ2 mRNA in HT-29 cells following treatments with ibuprofen or indomethacin……………………………………………………..
6.3.5 Expression of PPARα, PPARδ, PPARγ1 and PPARγ2 mRNA in HCT-116 cells following treatments with ibuprofen or indomethacin………………................................................
188 188 192 197 200
6.4 Discussion…………………………………………………………………...
203
Chapter 7 General Discussion……………………………………………...
209
Reference 217
List of tables
Page Table 1.1 A list of the main caspases and some of their substrates……… 20 Table 1.2 NSAID classification..…………………………………...………….. 34 Table 2.1 List of general chemicals and reagents…………………………... 47 Table 2.2 List of commercial kits and consumables………………………… 49 Table 2.3 Primer and probe sequences……………………………………… 50 Table 2.4 List of antibodies used for Western blotting analysis……..…….. 52 Table 2.5 Overview of the controls used in LDH assay…………………….. 69 Table 2.6 Composition of RIPA buffer………………………………………... 79 Table 3.1 The general characteristics of HCA-7, HT-29 and HCT-116 cell
lines used in this study……………………………………………... 116
Table 3.2 The EC50 value of ibuprofen and indomethacin determined in HCA-7 cell line……………………………………………………….
119
Table 3.3 The EC50 value of ibuprofen and indomethacin determined in HT-29 cell line………………………………………………………..
123
Table 3.4 The EC50 value of ibuprofen and indomethacin determined in HCT-116 cell line…………………………………………………….
126
Table 4.1 Potential biomarker molecules for apoptosis studies………….... 133 Table 4.2 Summary of the pattern of pro-caspase-8, -9, -3 and -7 protein
expression in HCA-7, HT-29 and HCT-116 cells following treatments with ibuprofen or indomethacin……………………....
150
Table 5.1 Comparison between the sequences of the COX-2, c-myc, β-catenin and TCF-4 cloned PCR products and the corresponding human mRNA sequence access from GeneBank…………………………………………………………….
160
Table 5.2 Summary of the pattern of COX-2, c-myc, β-catenin and TCF-4 mRNA expression in HCA-7, HT-29 and HCT-116 cells following treatments with ibuprofen or indomethacin…...……….
178
Table 6.1 Comparison between the sequences of the PPARα, PPARδ,
PPARγ1 and PPARγ2 cloned PCR products and the corresponding human mRNA sequence access from GeneBank……………………………….. ………………………….
190
Table 6.2 Summary of the pattern of PPARα, PPARδ, PPARγ1 and
PPARγ2 mRNA expression in HCA-7, HT-29 and HCT-116 cells following treatment with ibuprofen or indomethacin…...…..
204
List of figures
Page Figure 1.1 Classification of NSAIDs based on the relative specificity ratio
of COX-1 to COX-2 in human whole blood assay………………. 4
Figure 1.2 The transcriptional activation by PPARγ…………………………. 8
Figure 1.3 Hallmarks of the apoptotic and necrotic cell death processes…. 13
Figure 1.4 The putative apoptosis mechanism induced by PPARγ ligands.. 16
Figure 1.5 The mitochondrial-dependent apoptosis pathway…..…………... 18 Figure 1.6 Activation of pro-caspases……………………….......................... 21 Figure 1.7 The apoptosis signaling mechanism by Fas death receptor…… 26 Figure 1.8 The tumor necrosis factor receptor-1.....………..………………... 28 Figure 1.9 The signal transduction of survival signaling pathway………….. 32 Figure 1.10 The putative mechanism of action of NSAIDs in human CRC
cells in vitro……………………………………………….…………. 37
Figure 1.11 A summary of the experiment design…………………………….. 44 Figure 2.1 Determination of the optimal cell concentration…………………. 64 Figure 2.2 Determination of LDH activity of indomethacin..………………… 67 Figure 2.3 Determination of LDH activity of ibuprofen………………............ 68 Figure 2.4 Overview procedure of measuring LDH activity………............... 71 Figure 2.5 Apoptosis detection using M30 CytoDEATH assay...…………... 75 Figure 2.6 Maps of plasmid vectors used in this study……………………… 98 Figure 2.7 Maps of plasmid vectors used in this study……………………… 99 Figure 2.8 Maps of plasmid vectors used in this study……………………… 100 Figure 3.1 Principle of LDH assay………………………………………… 110 Figure 3.2 The sigmoid shape of dose response curve……………………... 111 Figure 3.3 Schematic representation of the determination of constant
EC50 value………………………………………………………….. 112
Figure 3.4 The typical epithelial appearance of a confluent monolayer of HCA-7, HT-29 and HCT-116 cells………………………………...
115
Figure 3.5 The cytotoxic effect of ibuprofen or indomethacin in HCA-7 cells…………………………………………………………………..
118
Figure 3.6 The cytotoxic effect of ibuprofen or indomethacin in HT-29 cells……….................................................................................
121
Figure 3.7 The cytotoxic effect of ibuprofen or indomethacin in HCT-116 cells…………………………………………………………………..
124
Figure 3.8 Schematic representation of the determination of constant EC50 values of ibuprofen or indomethacin in HCA-7, HT-29 and HCT-116 cells………………………………………………….
127
Figure 4.1 The percentage of apoptotic cells induced by ibuprofen or indomethacin in HCA-7 cells……...………………………………..
138
Figure 4.2 The percentage of apoptotic cells induced by ibuprofen or indomethacin in HT-29 cells………………………………………..
140
Figure 4.3 The percentage of apoptotic cells induced by ibuprofen or indomethacin in HCT-116 cells.…..............................................
142
Figure 4.4 Western blot analysis of caspase-8, -9, -3, -7 and β-actin expression in HCA-7 cells.……………………………..…………..
144
Figure 4.5 Western blot analysis of caspase-8, -9, -3, -7 and b-actin expression in HT-29 cells.………………………………...………..
146
Figure 4.6 Western blot analysis of caspase-8, -9, -3, -7 and b-actin
expression in HCT-116 cells.……………………………..……...... 148 Figure 5.1 Gel electrophoresis of PCR products……………………….......... 159 Figure 5.2 Standard curves........................................……………………….. 163 Figure 5.3 Electrophoresis of total RNA samples........……………………… 165
Figure 5.4 The linear regression plot of CT value of β-actin from HCA-7, HT-29 and HCT-116 cDNA samples………………………………
166
Figure 5.5 The modulation of COX-2, c-myc, β-catenin and TCF-4 mRNA expression in HCA-7 cells following treatment with ibuprofen or indomethacin…………………………………………………...........
168
Figure 5.6 The modulation of COX-2, c-myc, β-catenin and TCF-4mRNA expression in HT-29 cells following treatment with ibuprofen or indomethacin……........................................................................
172
Figure 5.7 The modulation of COX-2, c-myc, β-catenin and TCF-4 mRNA expression in HCT-116 cells following treatment with ibuprofen or indomethacin……………….....................................................
175
Figure 6.1 Gel electrophoresis PCR products…………………..……………. 189 Figure 6.2 Standard curves …………………………..………………………... 193
Figure 6.3 The modulation of PPARα, PPARδ, PPARγ1 and PPARγ2 mRNA expression in HCA-7 cells following treatment with ibuprofen or indomethacin….......................................................
194
Figure 6.4 The modulation of PPARα, PPARδ, PPARγ1 and PPARγ2 mRNA expression in HT-29 cells following treatment with ibuprofen or indomethacin...........................................................
198
Figure 6.5 The modulation of PPARα, PPARδ, PPARγ1 and PPARγ2 mRNA expression in HCT-116 cells following treatment with ibuprofen or indomethacin………………………………………….
201
The anticancer mechanism of ibuprofen atau indomethacin in
colorectal cancer cells
Abstract
Ibuprofen and indomethacin are among the frequently studied
non-steroidal anti-inflammatory drugs (NSAIDs) for their anticancer
activities. Besides being non-selective cyclooxygenase-2 (COX-2)
inhibitors, both NSAIDs are also direct ligands for peroxisome
proliferators-activated receptor-gamma (PPARγ). However, the precise
mechanism(s) of action whereby both NSAIDs exert their anticancer
effect remain unclear. In this study, we investigated the effects of both
NSAIDs in constitutively COX-2-expressing (HCA-7 and HT29) and non-
constitutively COX-2-expressing (HCT116) cell lines. Our initial aim of the
study was to determine the NSAID growth inhibitory effect as well as the
effective concentration to inhibit 50% of cell growth (EC50) of each NSAID
in each cell line using lactate dehydrogenase (LDH) release assay. The
apoptosis mechanism was then investigated using M30 CytoDEATH
assay prior to flow cytometry analysis. The apoptotic-related proteins
such as caspase-8, -9, -3 and -7 were also investigated using Western
blot analysis, whereas the modulation of mRNA expression of relevant
molecular targets such as COX-2, c-myc, β-catenin, TCF-4 and PPAR
subtypes (α, δ, γ1 and γ2) mRNA was quantified using Real-time PCR
analysis. Our results demonstrated that both NSAIDs produced
remarkable inhibition on the growth of all three cell lines tested. The
iii
inhibitory effect occurred in a concentration- and time-dependent
manner, with indomethacin (EC50 value >100 µM) being more potent
compared to ibuprofen (EC50 value >1000 µM). Furthermore, the ability
of both NSAIDs in inhibiting the growth of cells is likely not to be
associated with COX-2 expression. The evidence from M30 CytoDEATH
assay suggested that the major mode of cell death caused by both
NSAIDs was caspase-dependent apoptosis. This evidence was further
supported by Western blot analysis which indicated that the induction
occurred via caspase-9-dependent pathway, whereas the Real-time PCR
analysis showed that both NSAIDs appear to modulate gene expression
via a variety of different molecular targets in COX-2-dependent and/or
independent pathway(s) depending on the colorectal cancer (CRC) cell
type. However, alteration of TCF-4 and PPARγ1 mRNA expression are
likely essential for both NSAIDs to induce apoptosis. Thus, Wnt and
PPARγ signaling pathways may be involved in mediating the apoptosis
induced by both NSAIDs in CRC cells. In addition, PPARδ is found to be
another essential molecular target for indomethacin-induced CRC cell
apoptosis. In conclusion, our study may provide additional information
and evidence of the various mechanisms and actions of NSAIDs in
human CRC cells which may be useful in selecting effective apoptotic
drugs against specific CRC types. As we and others have shown, both
NSAIDs have anti-CRC activities and are potential anti-CRC agents.
Further studies on the effect of both NSAIDs on CRC cells remain
important, as they may be developed as chemotherapeutic agents for
human CRC.
iii
Antikanser mekanisme ibuprofen atau indomethacin dalam sel
kanser kolorektal
Abstrak
Ibuprofen dan indometasin adalah antara drug antiinflamatori
bukan steroid (NSAIDs) yang kerap dikaji untuk aktiviti-aktiviti anti
kansernya. Selain daripada perencat siklooksigenase-2 (COX-2) bukan
pilihan, kedua-dua NSAIDs ini adalah ligan langsung untuk reseptor-
gama pengaktif proliferators peroksimase (PPARγ).
Walaubagaimanapun, tindakan mekanisme atau mekanisme-mekanisme
yang tepat yang mana kedua-dua NSAIDs mengenakan kesan anti
kanser masih lagi tidak jelas. Kajian ini menyiasat kesan garis sel pada
kedua-dua NSAIDs dalam sebahagian ekspresi COX-2 (HCA-7 dan
HT29) dan bukan pada sebahagian ekspresi COX-2. Pada awalnya,
kajian ini dijalankan untuk menentukan kesan perencatan pertumbuhan
NSAIDs dan juga kepekatan (concentration) yang berkesan untuk
merencat 50% daripada sel pertumbuhan (EC50) pada setiap NSAIDs di
setiap garis sel menggunakan dehidrogenase laktat (LDH) yang
mengeluarkan assay. Mekanisme apoptosis kemudiannya disiasat
menggunakan assay M30 CytoDEATH sebelum analisis aliran sitometri
dijalankan. Protein-protein berkaitan apoptotic seperti caspase-8, -9, -3
dan -7 juga telah disiasat menggunakan analisis blot Western, manakala
modulasi ekspresi mRNA untuk molekul sasaran yang berkaitan seperti
COX-2, c-myc, β-catenin, TCF-4 dan subjenis-subjenis PPAR mRNA (α,
iii
δ, γ1 dan γ2) dikuantitikan menggunakan analisis PCR masa-sebenar
(REAL-time). Keputusan menunjukkan bahawa kedua-dua NSAIDs
menghasilkan perencatan yang luar biasa pada pertumbuhan ketiga-tiga
garis sel. Indometasin (nilai EC50 > 100 µM) adalah lebih kuat jika
dibandingkan dengan ibuprofen (nilai EC50 > 1000 µM). Tambahan pula
kebolehan kedua-dua NSAIDs dalam merencat pertumbuhan sel-sel
mungkin tidak akan dikaitkan dengan ekspresi COX-2. Bukti dari assay
M30 CytoDEATH mengusulkan bahawa mod utama kematian sel adalah
disebabkan kedua-dua NSAIDs adalah apoptosis yang bergantung pada
caspase. Bukti ini seterusnya disokong oleh analisis blot Western yang
menunjukkan bahawa cetusan berlaku melalui laluan yang bergantung
pada caspase-9, manakala analisis PCR masa-sebenar (REAL-time)
menunjukkan bahawa kedua-dua NSAIDs kelihatan memodulat ekspresi
gen melalui pelbagai sasaran molekul yang berbeza dalam laluan atau
laluan-laluan yang bergantung atau tidak bergantung pada COX-2 dan
ini bergantung kepada jenis sel kanser kolorektal (CRC).
Walaubagaimanapun perubahan pada TCF-4 dan ekspresi mRNA
PPARγ adalah agak penting kepada kedua-dua NSAIDs untuk mencetus
apoptosis. Oleh itu laluan-laluan isyarat Wnt dan PPARγ mungkin terlibat
sebagai perantara apoptosis yang dicetus oleh kedua-dua NSAIDs
dalam sel-sel CRC. Sebagai tambahan, PPARδ pula didapati sebagai
satu lagi sasaran molekul yang penting untuk apoptosis sel CRC
cetusan-indometasin. Kesimpulannya, kajian ini mungkin dapat memberi
maklumat tambahan dan bukti pelbagai mekasisme-mekanisme dan
tindakan-tindakan NSAIDs dalam sel CRC manusia, yang mungkin
iii
berguna untuk memilih drug apoptotic yang berkesan terhadap jenis
CRC yang spesifik. Seperti yang telah kami dan penyelidik-penyelidik
lain telah tunjukkan, kedua-dua NSAIDs mempunyai aktiviti-aktiviti anti-
CRC dan berpotensi sebagai agen anti-CRC. Kajian-kajian lanjut
mengenai kesan kedua-dua NSAIDs pada sel-sel CRC tetap penting
kerana mereka boleh dibangunkan sebagai agen kemoterapeutik untuk
CRC manusia.
Chapter 1
Introduction
1.1 Introduction of NSAIDs
Non-steroidal anti-inflammatory drugs (NSAIDs) were first
introduced in the 1900s, and have subsequently been widely used to
treat inflammation, mild-to-moderate pain and fever (Vane and Botting,
1998). Although they have been used for more than two decades and
their safety has constantly been reviewed, they have only recently been
proposed as anti-cancer agents (Vane and Botting, 1998). NSAIDs work
mainly by inhibiting cyclooxygenase (COX) and preventing the formation
of prostaglandins (PGs) (Smith and Goh, 1996; Badawi et al., 2000; Han
et al., 2001). There are two isoforms of COX which are known as COX-1
and COX-2 (Sheng et al., 1997; Badawi et al., 2000). COX-1 is
continuously secreted within the stomach and duodenum where it helps
maintain healthy stomach lining, normal kidney function and the clotting
action of blood platelets (Sheng et al., 1997). In contrast, the COX-2
enzyme is primarily found at sites of inflammation (Sheng et al., 1997). It
is induced by inflammatory stimuli such as interleukin-1, and is
suppressed by glucocorticoids (Sheng et al., 1997).
Blocking of COX-2 is effective in relieving pain and inflammation,
whereas inhibiting of COX-1 often produces unacceptable
gastrointestinal side effects such as diarrhea, bloating, heartburn,
stomach upset and ulcers (Vane and Botting, 1998). Various
conventional NSAIDs such as aspirin, diclofenac, ibuprofen,
indomethacin, naprosyn and piroxicam are being used as anti-cancer
agents (Soh and Weinstein, 2003). These NSAIDs inhibit both COX-1
and COX-2 to differing degrees in terms of the relative specificity ratio
(IC50; Figure 1.1) (Mitchell et al., 1994; Bishop-Bailey et al., 1997;
Bishop-Bailey and Warner, 2003).
The lower ratio value reflects the more potent activity of NSAIDs
against COX-2 inhibition. Thus, SC58125 (celecoxib derivative) and
celecoxib appear to be more selectively involved in COX-2 inhibition
activity than naproxen and flurbiprofen (Figure 1.1). Besides indicating
the selectivity of NSAIDs in COX-2 inhibition, ratio IC50 adequately
explains the variations in the side effects of NSAIDs at their anti-
inflammatory doses (Vane and Botting, 1998). Selective COX-2 inhibitors
such as celecoxib which have higher potency on COX-2 inhibition activity
will have potent anti-inflammatory activity with fewer side effects on the
stomach and kidney (Henry, 2003; Langman, 2003; Wolfe, 2003),
whereas non-selective COX-2 inhibitors such as aspirin, piroxicam,
ibuprofen and indomethacin which have a higher potency against COX-1
than against COX-2 inhibition activity, were found to produce high
gastrointestinal toxicity and cause the most damage to the stomach at
inflammatory doses (Lanza et al., 1989; Rodriguez et al., 1994).
1.2 The non-selective COX inhibitors
The ability of NSAIDs to decrease the COX-1/COX-2 expression
has allowed consideration of NSAIDs as potential anti-cancer agents for
cancer treatment including human colorectal cancer (CRC) treatment
(Vane and Botting, 1998). COX-2 expression has been shown to be up-
regulated in human CRC when compared with normal adjacent colonic
mucosa (Na and Surh, 2003). Inhibition of the COX-2 expression is
important because over expression of COX-2 provides tumor cells with
growth and survival advantages including resistance to apoptosis, and
increased invasiveness or angiogenesis (Mohammed et al., 1999;
Cianchi et al., 2001; Yamada et al., 2001; Na and Surh, 2003).
Thus, targeting the inhibition of COX-2 is regarded as an affective
and promising strategy for cancer prevention and treatment (Han et al.,
2001; Na and Surh, 2003). Indeed, the expression of COX-2 is
stimulated by growth factors and tumor promoters, and it is often
selectively expressed in tumors cells including CRC cells (Eberhart et al.,
1994; Sano et al., 1995; Han et al., 2001). Thus, the expression of COX-
2 is believe that plays an important role in cancer, and is often correlated
with tumorigenesis. This correlation was supported by the finding that in
two mutated adenomatous polyposis coli (APC) background mice where
the COX-2-/- mice developed fewer intestinal polyps than the COX-2+/+
mice (Oshima et al., 1996; Han et al., 2001).
Many epidemiologic and clinical studies have revealed that regular
ingestion of NSAIDs such as aspirin, sulindac and indomethacin can
reduce the risk of developing several malignant tumors including CRC
(Giardiello et al., 1995; Giovannucci et al., 1995; Boolbol et al., 1996).
Based on the evidences above, it is logical to choose a selective COX-2
inhibitor that is as effective as non-selective inhibitors and could prevent
PGs formation in inflamed areas without interfering with the activity in
stomach and kidney (Vane and Botting, 1998). However, COX-2 appears
to have multiple functions and has been shown to be important in
ovulation and vasoprotection, and is essential for normal kidney
development (Dahl, 1999). Indeed, both COX-1 and COX-2 are
expressed in inflamed joints, so it is important that mixed COX inhibition
is required for the maximal anti-inflammatory effects (Dahl, 1999).
1.3 Evaluation of NSAIDs as PPARγ ligands
Non-selective COX inhibitors are regarded as effective and
promising anti-cancer agents for cancer prevention or treatment.
However, the fact that NSAIDs retain the anti-proliferative activity in a
variety of cells that do not express COX-2 would argue against the
relevance of COX-2 inhibition as a target for NSAIDs in cancer cells
(Hanif et al., 1996; Zhang et al., 1999; Chapple et al., 2000; Smith et al.,
2000; Han et al., 2001; Hull et al., 2003). Indeed, the concentration of
NSAIDs required to induce cell death was several orders of magnitude
higher than that required to inhibit the activity of COX (Mitchell et al.,
1993; Han et al., 2001). For example, the IC50 ratio of indomethacin to
inhibit COX-1 and COX-2 in intact cells were reported to be 28 nM and
1.7 µM, respectively (Mitchell et al., 1993), whereas the concentration of
indomethacin used to induce cell death in the same cells was 500 µM
(Zhang et al., 1999). Therefore, the anti-proliferative activity of NSAIDs
would seem to occur via COX-independent mechanisms (Hull et al.,
2003; Soh and Weinstein, 2003).
One possible mechanism was suggested to be via the peroxisome
proliferators-activated receptor-gamma (PPARγ), a ligand-activated
transcription factor pathway (Smith et al., 2000; Hull et al., 2003; Soh and
Weinstein, 2003). According to Lehmann et al. (1997), several NSAIDs
including ibuprofen and indomethacin, can interact directly and
specifically bind with PPARγ ligand binding domain (LBD). Upon ligand
binding, PPARγ becomes activated and heterodimerises with retinoid X
receptor (RXR) (Figure 1.2) (Boitier et al., 2003). The co-repressors will
then dissociate from the complex which leads to an active PPARγ/RXR
complex that binds to the gene regulatory sites on DNA termed PPAR
response elements (PPRE). There is also evidence of the involvement of
co-activator proteins that enhance the activity of the PPARγ/RXR
complex. Binding of the PPARγ/RXR complex to PPRE increases the
transcription of numerous genes including genes involve in
tumorigenesis (Boitier et al., 2003). The ability of NSAIDs to bind to
PPARγ has been previously assessed in a competition-binding assay
using [3H] BRL49653 and bacterially expressed PPARγ LBD (Lehmann
et al., 1997). Though not all NSAIDs activate PPARγ through direct
interactions with the receptor, the fact that NSAIDs bind and thereby
activate PPARγ activity, subsequently define them as the PPARγ ligands
(Lehmann et al., 1997).
Interestingly, ibuprofen and indomethacin used in this study are
non-selective COX inhibitors and direct PPARγ ligands. Activation of
PPARγ by ibuprofen and indomethacin was the strongest of any of the
NSAIDs that were evaluated in CRC (Nixon et al., 2003). Furthermore,
both NSAIDs may activate more than one PPAR subtypes. For example,
ibuprofen activates PPARdelta (PPARδ) and PPARγ (Kojo et al., 2003),
whereas indomethacin shows agonistic activity for PPARalpha (PPARα)
and PPARγ (Lehmann et al., 1997) or PPARγ alone based on two
different studies (Kojo et al., 2003). As dual ligands have been proven to
be more effective at preventing colorectal carcinogenesis, ibuprofen and
indomethacin are therefore potential anti-cancer agents for human CRC.
1.4 The role of PPARγ ligands in cancer
The mechanisms of anti-cancer effect by the PPARγ ligands have,
however, not been fully elucidated. In general, activation of PPARγ by its
ligand is associated with cancer prevention (Brockman et al., 1998;
Sarraf et al., 1998; Kitamura et al., 1999). The PPARγ ligands have anti-
proliferative and pro-differentiation effects in slowing the proliferation of
tumor cells including breast (Elstner et al., 1998; Rubin et al., 2000),
colon (Sarraf et al., 1998), prostate (Kubota et al., 1998; Hisatake et al.,
2000; Shappell et al., 2001), gastric (Takahashi et al., 1999) and
pancreatic (Motomura et al., 2000) cancer cells.
In support of the former findings, the thiazolidinediones (TZD)
group of PPARγ ligands has been shown to inhibit proliferation of breast,
prostate and colon cells (Elstner et al., 1998; Kubota et al., 1998; Sarraf
et al., 1998). In contrast, the ligands promote the development of colon
tumors in transgenic mice in which one copy of the gene encoding the
APC is knocked out, predisposing them to colon cancer (Lefebvre et al.,
1998). However, another study demonstrated that treatment of patients
with liposarcomas with a PPARγ ligand results in anti-neoplastic pro-
differentiation which may decrease the proliferative rate of the cancer
cells and slow the progression of the disease (Demetri et al., 1999).
There are also observations that PPARγ ligands induce growth arrest
and the synthesis of differentiation markers in human CRC cells
(Kitamura et al., 1999). Furthermore, PPARγ ligands have been shown to
be potent inhibitors of angiogenesis, a process necessary for solid-tumor
growth and metastasis (Xin et al., 1999). Although much of the evidence
suggests that activation of PPARγ by its ligands could promote the
suppression of tumor growth and inhibit carcinogenesis, the precise
mechanisms involved remain unclear.
PPARγ ligands have been shown to exert their anti-cancer cell
proliferation through the induction of apoptosis (Yang and Frucht, 2001;
Clay et al., 2002). Although the precise role of PPARγ in cancer cells is
still debatable, accumulating evidence has reported that PPARγ ligands
inhibiting cancer cell growth via apoptosis (Yang and Frucht, 2001; Clay
et al., 2002; Na and Surh, 2003). The apoptosis pathway is currently the
key mechanism by which anti-cancer agents kill tumor cells. The ability of
PPARγ ligands to decrease COX-2 expression and initiate apoptosis has
made them to be considered as potential therapeutic agents for cancer
treatment (Yang and Frucht, 2001; Debatin, 2004). Therefore,
identification of the molecular pathways induced by PPARγ ligands will
be of both basic and clinical importance.
1.5 Apoptosis
Apoptosis, first reported in 1964, is also known as programmed
cell death (Gewies, 2003). It is the physiological process by which
unwanted cells are eliminated during development and other normal
biological processes (Gewies, 2003). Moreover, cytotoxic T-lymphocytes
(CTLs) kill virus-infected cells are also via the induction of apoptosis
(Roberts, 2000). Currently, understanding of the apoptosis plays an
important role in the study of physiological processes, particularly those
involved in cancer research (Roberts, 2000). For example, the mutation
of the p53 gene produces defective proteins that are often found in
cancer cells which contribute to resistance to apoptosis (Debatin, 2004).
Moreover, immunotherapy of tumors requires target cells with an
apoptosis-sensitive phenotype (Debatin, 2004).
Apoptosis is of major importance in the pathogenesis of several
diseases including cancer, AIDS and neurological disorders such as
Alzheimer’s and Parkinson’s diseases (Roberts, 2000). It is believed that
defects in the apoptotic pathways represent hallmarks of tumorigenesis
(Okada and Mak, 2004). Recent research on new cancer therapies has
therefore focused on devising ways to overcome this resistance and to
trigger apoptosis of cancer cells (Okada and Mak, 2004).
1.6 Morphological features of necrosis and apoptosis
In the cell death process, necrosis occurs when cells are exposed
to a serious physical or chemical insult (Gewies, 2003). The necrotic
mode of cell death, in which cells suffer a major insult, results in a loss of
membrane integrity, swelling and cell rupture (Figure 1.3). During
necrosis, the cellular contents are released uncontrolled into the
environment which results in damage to surrounding cells and a strong
inflammatory response in the corresponding tissue (Gewies, 2003).
In contrast to necrosis, apoptosis is characterised by typical
morphological and biochemical hallmarks including cell shrinkage,
nuclear DNA fragmentation and membrane blebbing (Debatin, 2004).
The morphological changes such as cell shrinkage and deformation
results in the cells loss of contact with neighboring cells and chromatin
condensation (Figure 1.3). The chromatin is then localised at the nuclear
membrane, the plasma membrane undergoes blebbing and finally the
cell is fragmented into compact membrane-enclosed structures termed
apoptotic bodies. The apoptotic bodies contain cytosol, condensed
chromatin and organelles. They are removed from the tissue by
phagocytosis or secondary necrosis without causing an inflammatory
response.
1.7 The putative mechanisms of PPARγ ligand-induced apoptosis
As described above, treatment with PPARγ ligands has been
widely implicated to reduce the cancer cell growth rate and induce cell
terminal differentiation of a number of human cancers including CRC via
induction of apoptosis (Yang and Frucht, 2001; Boitier et al., 2003).
According to Goke et al. (2000), the induction of apoptosis by PPARγ
ligands is increased by co-stimulation with tumor necrosis factor (TNF)-
related apoptosis-inducing ligand (TRAIL), a member of the TNF family.
Furthermore, inhibition of cell growth observed in human breast cancer
cells treated in vitro with PPARγ ligands is accompanied with a marked
increase in apoptosis and a profound decrease of Bcl-2 gene expression
(Elstner et al., 1998). In addition, troglitazone-induced apoptosis has
been demonstrated to be associated with caspase-dependent and -
independent mechanisms (Yoshizawa et al., 2002). More importantly,
Yoshida et al. (2003) recently proposed the mechanisms concerning
PPARγ ligand-induced apoptosis would be occurring via a) perturbation
of mitochondrial membrane permeability; b) binding of death receptor to
its ligand; and c) phosphatidylinositol-3-kinase (PI3)-serine/threonine
kinase (Akt)-apoptosis protease activating factor-1 (Apaf-1) system
(down-regulation of survival signaling pathway) (Figure 1.4).
1.7.1 Mitochondrial-dependent apoptosis
PPARγ ligands mainly induce apoptosis via perturbation of inner
mitochondria membrane permeability (Yoshida et al., 2003). This
mitochondrial-dependent apoptosis was termed intrinsic apoptosis where
signals from various stimuli disturb the membrane permeability of the
mitochondria and subsequently release the apoptogenic factor such as
cytochrome c into the cytoplasm (Yoshida et al., 2003). During intrinsic
apoptosis, anti-apoptotic proteins such as B-cell lymphoma 2 (Bcl-2) and
the pro-apoptotic protein such as Bcl-2 associated X protein (Bax) are
induced to oligomerise and migrate from the cytoplasm to the
mitochondria escort by various BH3-Only-Protrein (Debatin, 2004). Once
the protein is inserted into the outer mitochondria membrane, it induces
cytochrome c release by the creation or alteration of the mitochondria
membrane pores (Debatin, 2004).
In the cytoplasm, cytochrome c combines with Apaf-1 and pro-
caspase-9 to form a complex termed apoptosome, in the presence of
adenosine 5’-triphosphate (ATP) to activate caspase-9 (Figure 1.5)
(Roberts, 2000; Gewies, 2003; Debatin, 2004). Caspase-9 subsequently
activates downstream executor caspases such as caspase-3, -6 and -7
(Roberts, 2000). Activation of caspase-3 and the subsequent degradative
events are probably triggered to initiate the apoptosis (Roberts, 2000;
Yoshida et al., 2003; Debatin, 2004). In most PPARγ ligands induced
apoptosis cases, caspase-3 is the final molecule where activation of
caspase-3 will then causes DNA fragmentation (Yoshida et al., 2003).
However, the mitochondrial pathway must not always associated
with caspase activation (Debatin, 2004). The mechanism varies
depending on the release of apoptogenic factor such as cytochrome c,
Smac/Diablo or apoptosis inducing factor (AIF) (Figure 1.5) (Debatin,
2004). The release of cytochrome c triggers caspase-9 activation through
the formation of cytochrome c/Apaf-1/caspase-9 in the presence of ATP
to form an active apoptosome complex as described above (Roberts,
2000; Gewies, 2003; Debatin, 2004). Thus, the release of cytochrome c
from mitochondria always reflects the mitochondrial→caspase-
9→executor caspase activation→apoptosis (Cai et al., 2002; Debatin,
2004). The Smac/Diablo promotes caspase activation by neutralising the
inhibitory effects of inhibitors of apoptosis proteins (IAPs) and this may
also imply the same caspase activation pathway (Debatin, 2004).
However, AIF causes DNA condensation or initiate apoptosis without
activating any caspases (Debatin, 2004).
1.7.1.1 Caspase activation in apoptosis
Caspase(s) with the generic name c denotes a cysteine protease
and aspase refers to the aspartate specific cleaving ability of these
enzymes (Gewies, 2003). The individual members are then numbered
according to their chronological order of publication (Robert, 2000).
Caspase activation is recently demonstrated as central importance
(hallmark) in the apoptosis signaling network as well as DNA
fragmentation, which is activated in most cases of apoptotic cell death
(Roberts, 2000; Gewies, 2003; Debatin, 2004). To date, 14 different
members of the caspase have been described in mammals with
caspase-11 and -12 only identified in mouse (Gewies, 2003). In broad
terms, there are two groups of caspases; initiator caspase (such as -2, -
8, -9, and -10) and executor caspase (such as -3, -6 and -7) (Table 1.1)
(Roberts, 2000; Gewies, 2003; Debatin, 2004).
In the cell, caspases are synthesised as inactive zymogens. The
inactive form of initiator caspase (e.g caspase-8) has longer domain than
inactive form of executor caspase (e.g caspase-3) (Figure 1.6 A)
(Gewies, 2003). The inactive form of caspase is so-called pro-caspase
which at the N-terminus carry a pro-domain followed by a large and a
small subunit. The large and the small subunits are sometimes separated
by a linker peptide. Upon maturation, the pro-caspase is proteolytically
processed between the large and small subunit where the domain is
removed resulting in forming a heterotetramer consisting of two small
and two large subunits. The heterotetramer is an active form of caspase.
The disappearance of pro-caspase bands in Western blot analysis
reflects processing of the zymogen to generate the active form of the
specific caspase (Gewies, 2003; Cheah and Azimahtol, 2004). After the
initiator pro-caspase is cleaved and activated, it subsequently actives the
corresponding downstream executor pro-caspase molecules to become
active caspase which in turn initiating the apoptosis (Figure 1.6 B)
(Gewies, 2003).
The expression level of individual caspase may have an impact on
their overall activity, since deficient expression level may simply impair
caspase activation (Debatin, 2004). For example, MCF-7 breast
carcinoma cells completely lack caspase-3 expression due to a frame
shift mutation within exon 3 of the caspase-3 gene (Janicke et al., 1998).
These cells can be sensitised to treatment with cytotoxic drugs by
transfection of pro-caspase-3. Alternatively, caspase-8 expression was
found to be frequently inactivated by hypermethylation of regulatory
sequences of the caspase-8 gene in a number of different tumor cells
derived from neuroblastoma, malignant brain tumors, Ewing tumors and
small-cell lung carcinoma (Teitz et al., 2000; Fulda et al., 2001).
Importantly, restoration of caspase-8 expression by gene transfer or
demethylation treatment sensitised resistant tumor cells to death
receptor-induced or drug-induced apoptosis (Fulda et al., 2001). In
contrast, gene knockout experiments targeting caspase-9 and -3 resulted
in mortality as a result of severe defects in brain development (Kuida,
1998). Moreover, caspase-8-deficient embryos died after day 12
(Varfolomeev, 1998). This and the observation indicating that cell lines
derived from such knockout experiments are resistant to distinct
apoptosis stimuli underlines the importance of caspases as pro-apoptotic
mediator (Gewies, 2003). Indeed, it has been recognised that caspases
play an important role in the apoptotic signaling machinery (Earnshaw,
1999).
Given the important role of caspases as the final effectors
molecule of apoptosis, the ability of anti-cancer agents such as PPARγ
ligands to trigger caspase activation appears to be a critical determinant
of sensitivity or resistance to cytotoxic therapies (Debatin, 2004). Indeed,
caspases are useful biomarkers for apoptosis, as outlined below
(Roberts, 2000). As a consequence, determination of caspase activation
in various forms of cell death including PPARγ ligand-induced apoptosis
may be an important factor in cancer chemotherapies (Debatin, 2004).
1.7.2 Death receptor-ligand apoptosis
The death receptor-ligand system is so called extrinsic apoptosis
because the apoptosis signal is initiated by the death receptor at cell
surface (Goke et al., 2000; Gewies, 2003; Debatin, 2004). The death
receptor belongs to the TNF gene superfamily and generally has several
functions other than initiating apoptosis (Debatin, 2004). The best-
characterised death receptors are CD95 (or Fas), TNF receptor-1 (TNF-
R1) and TRAIL receptor (Goke et al., 2000; Roberts, 2000). The PPARγ
ligand-induced death receptor apoptosis is at all times associated with
the TNF receptor family (Goke et al., 2000).
1.7.2.1 Signaling by CD95/Fas
According to Ashkenazi and Dixit (1998), there are three main
roles of CD95 signaling:
1. mediate killing of cells by T-cells e.g. CTL-mediated killing of virus-
infected cells;
2. mediate deletion of activated T-cells at the end of an immune
response;
3. mediate destruction of inflammatory and immune cells in immune-
privileged sites.