Cancer Prevention: Dietary Factors and Pharmacology

Dedication......Page 6
Preface......Page 8
Contents......Page 12
Contributors......Page 14
1 Introduction......Page 17
2.1 Computer Hardware......Page 19
2.2.6 CHARMM (Chemistry at HARvard Macromolecular Mechanics)......Page 20
2.3.6 Ligplot......Page 21
2.4.2 Reagents......Page 22
3.1 Computational Strategies......Page 23
3.1.1 Virtual Screening......Page 25
3.1.2 Molecular Docking [ 26 ]......Page 26
3.1.4 Shape-Similarity Screening Method [ 26 ]......Page 27
3.2 Summary of Computational Strategies......Page 28
3.3.4 Anchorage-­Independent Neoplastic Transformation Assay......Page 29
3.3.7 Lentiviral Infection......Page 30
3.3.10 Cell Cycle Assay......Page 31
3.3.12 Preparation of Compound of Interest—Sepharose 4B Beads and Pull-Down Assay......Page 32
3.3.14 In Vitro PI3-K Kinase Assay......Page 33
3.4.1 Solar UV-Induced 2 Stage Skin Carcinogenesis Model......Page 34
Acute Solar UV Experiments......Page 35
3.4.2 In Vivo Xenograft Mouse Model......Page 36
General Methods......Page 37
Injection of Tumor Cells......Page 38
General Methods......Page 39
4 Notes......Page 40
5 Conclusions......Page 43
References......Page 44
1.1.1 Cell Proliferation or Viability Assay......Page 49
1.1.3 Cellular Apoptosis Analysis Using Annexin V/FITC......Page 50
1.1.5 Cellular Apoptosis Analysis by Detection of Caspase Activities and Levels of Cleaved Caspases......Page 51
1.2.2 Immunofluo-rescence Analysis......Page 52
1.2.4 Chromatin Immunoprecipitation (ChIP) Assay......Page 53
2.1.6 Chromatin Immunoprecipitation Assay......Page 54
2.2.6 Immunoassay for Discovering Kinase Inhibitors......Page 55
2.2.9 Chromatin Immunoprecipitation Assay......Page 56
3.2 Cell Cycle Analysis......Page 57
3.5 Analysis of Apoptosis by Caspase Assay......Page 58
3.6.3 Assay Procedure......Page 59
3.7.1 Summary and Introduction......Page 60
3.8.1 Summary and Introduction......Page 61
3.8.3 Determining the Protein Concentration (BCA Method)......Page 62
3.9.2 Cell Fixation, Chromatin Isolation, and Sonication Shearing......Page 63
3.9.4 Addition of Antibodies to Pre-cleared Chromatin......Page 64
4 Notes......Page 65
References......Page 67
1 Introduction......Page 69
Equipment......Page 71
Reagents and Solutions......Page 72
ARE-Luciferase Activity Induction in HepG2-C8 Cells......Page 73
Detoxifying/Antioxidant Enzymes Assay in HepG2-C8 Cells......Page 74
Notes......Page 75
3 Animal Model Approaches for Chemoprevention Studies Based on NRF2 Targeting......Page 76
DMBA-TPA Induced Skin Carcinogenesis......Page 77
Dextran Sulfate Sodium (DSS)-Induced Experimental Colitis......Page 78
Reagents and Solutions......Page 79
Plasma and Bioanalytical Analysis......Page 80
Pharmacokinetic/Pharmacodynamic (PK/PD) Model Development......Page 81
4 Epigenetics Approaches to Study NRF2 Regulation......Page 83
Reagents and Solutions......Page 84
Bisulfite PCR Amplification......Page 85
TA Cloning of Target DNA Fragment......Page 86
Equipment......Page 87
Reagents and Solutions......Page 88
Collecting and Cross-­linking Adherent Cells......Page 89
Shearing the Chromatin into ~200–500 bp Fragments......Page 90
Washing the Bound Chromatin......Page 91
Purifying the DNA ( Note 18)......Page 92
Data Analysis......Page 93
5 Notes......Page 94
References......Page 96
1.1 Prostate Cancer Chemoprevention......Page 100
1.2 Cancer Chemopreventive Efficacy of Silibinin......Page 101
1.3 Prostate Cancer Biomarkers Employed in Establishing the Chemopreventive Efficacy of Silibinin......Page 102
2.1 Equipment......Page 103
2.2 Reagents and Solutions......Page 104
3.2 Cytosolic and Nuclear Fraction Preparation from Cell Cultures......Page 106
3.4 Cytosolic and Nuclear Fraction Preparation from Prostate Tumor Tissue......Page 107
3.5 Protein Estimation......Page 108
3.6.2 Electrophoresis......Page 109
3.6.5 Enhanced Chemiluminescence (ECL)......Page 110
3.7 Immunofluo-rescence in Cell Cultures......Page 111
3.8.1 Paraffin Tissue Processing......Page 112
3.9.2 Antigen Retrieval......Page 113
3.9.3 Immunofluo-rescence......Page 114
3.10.4 Counterstaining......Page 115
3.10.6 Microscopic Observation and Analysis......Page 116
5 Conclusions......Page 117
References......Page 118
1 Introduction......Page 121
2 Approach Used to Evaluate the Chemopreventive Activity of BRBs (Fig.  1)......Page 123
3.1 Producing and Standardizing Black Raspberry Powder......Page 124
3.2 Testing for Toxicity in Rodents......Page 127
3.3 Evaluating Anticarcinogenic Effects in Animals and Mechanisms of Action......Page 128
3.3.1 Rat Esophagus Model of Squamous Cell Carcinoma......Page 129
Quantitative Immunohistochemistry......Page 132
Western Blot Analysis......Page 133
Some Results of Mechanistic Studies in the Rat Esophagus Model......Page 134
3.4.1 Hexane Extract......Page 135
3.4.3 Ethanol–H 2 O: Insoluble (Residue) Fraction......Page 136
3.4.5 Freeze-Drying of the Ethanol–H 2 O (80:20)-Soluble Extract and Sugar Fractions......Page 137
3.4.6 Testing the BRBs and Their Component Extracts/Fractions for Anticarcinogenic Activity in the Rat Esophagus Model......Page 138
4.2 “Pilot” Trial of BRBs for Chemopreventive Efficacy in Patients with Barrett’s Esophagus......Page 140
4.3 Phase II Trial of Strawberries (STRW) for Chemopreventive Efficacy in Chinese Patients with Esophageal Dysplasia......Page 142
References......Page 145
1 Introduction......Page 148
2.2 Selection of Mice (Table  2)......Page 152
2.3.2 Genotyping for ULp53 Mice (Fig.  1)......Page 153
Genotyping for Rb1 F19/F19 ; Trp53 F2-10/F2-10 Mice......Page 155
3.1 General Protocols and Tumor Phenotyping Methods for Animal Studies......Page 156
3.2.1 A/J Mouse Lung Adenoma Model Induced by Tobacco Specific Carcinogens such as B(a)P and NNK......Page 157
3.2.2 The A/J-p53 Transgenic Mouse and Wild Type Littermate Lung Adenocarcinoma Model Induced by VC......Page 159
3.3 Lung SCC Mouse Model......Page 160
3.4 SCLC Mouse Model......Page 163
References......Page 164
1 Introduction......Page 167
2.1 Equipment......Page 169
2.2 Reagents and Solutions......Page 170
3.1 Animal Care......Page 172
3.3 Long-Term Experiment to Induce Adenocarcinomas......Page 173
3.4 Preparation of the Colon for Macroscopic Examination......Page 175
3.5.1 Prepare the Paraffin-Embedded Tissue Sections......Page 176
3.6 Immunohisto-chemical Analysis......Page 177
3.6.2 Antigen Unmasking......Page 178
3.7 Immunoblot Analysis of Protein Expression......Page 179
3.7.3 Immunoblot Analysis......Page 180
3.8   EMSA......Page 181
3.8.2 The Nucleic Acid Protein Binding Reaction......Page 182
References......Page 183
1 Introduction......Page 185
1.1 Principle of the Assays......Page 186
1.2 Advantages and Disadvantages of the Assays......Page 187
2.2 Reagents and Solutions......Page 188
3.1.1 Cell Culture......Page 189
3.1.4 Preparing the Injection Compounds and Loading the Sensor Cartridge......Page 190
3.1.7 Data Analysis......Page 191
3.2.1 Preparing the Plate for the Steady-State ATP Measurements......Page 193
3.2.3 Data Analysis......Page 194
4 Notes......Page 195
5 Summary......Page 197
References......Page 198
1 Introduction......Page 200
2.1 Equipment......Page 203
2.3 Reagents and Solutions......Page 204
3.2 Cell Treatments......Page 206
3.4 Extracting RNA......Page 207
3.5 Preparation of Poly(A) + RNA......Page 208
3.6 Creating cDNA from RNA......Page 210
3.9 Perform End Repair of cDNA Fragments......Page 211
3.12 PCR Amplification......Page 212
4 Notes......Page 213
References......Page 214
1 Introduction......Page 216
2.1 Cells and Tissues......Page 217
2.2.1 Keratinocyte Harvesting Supplies......Page 218
2.2.3 3T3 Culturing Supplies......Page 219
2.2.6 DNA and RNA Supplies......Page 220
2.3.1 General Cell Culture Laboratory Reagents......Page 221
2.3.3 Keratinocyte Media......Page 222
2.3.5 Medium for 3T3 Culture......Page 223
2.5.1 Reagents for Preparing DNA and RNA from Epidermal Keratinocytes......Page 224
3.2.1 Keratinocyte Harvest and Seeding......Page 225
Analysis of Mass Cultures......Page 227
3.3.2 Subculturing the 3T3 Cell Line......Page 232
3.3.4 Irradiation and Seeding of the 3T3 Feeder Layer......Page 233
3.3.6 Analysis of Clonal Cultures (Counting and Measuring Colonies)......Page 234
3.4.1 Preparation of Keratinocyte Cells......Page 235
3.4.2 CD34 and CD49f (Alpha6 Integrin) Staining......Page 238
3.4.4 Analysis of Cell Cycle......Page 239
3.4.5 Interpreting Scatterplots......Page 240
3.5.2 Preparation of RNA......Page 241
3.5.4 Preparation of RT-PCR......Page 243
3.5.5 Analysis of RT-PCR......Page 244
4 Notes and Troubleshooting......Page 245
References......Page 247
1 Introduction......Page 248
2.1 Equipment......Page 251
3.2 DNA Isolation......Page 252
3.4 DNA Hydrolysis......Page 253
3.5.2 Oasis MAX......Page 254
3.8 LC-ESI-MS/MS-SRM Analysis......Page 255
4 Notes......Page 257
References......Page 258
1 Introduction......Page 260
2.2 Reagents and Solutions......Page 262
3.3 Immunostaining with Multiple Primary and Secondary Antibodies......Page 263
3.4.1 Antibody Specificity......Page 265
3.4.4 Co-localization and Visualization of Multiple Fluorescent Biomarkers in a Single Section......Page 266
3.4.5 Quantifying Density/Expression of Select Biomarker/Structures in Tissue......Page 268
4 Notes......Page 270
References......Page 275
1 Introduction......Page 276
2.1 Improving Agents......Page 278
2.2 Identifying Better Biomarkers......Page 283
2.3 Building and Enhancing Cohorts......Page 284
2.4 Improving Elements of Overall Trial Design......Page 285
2.5 Establishing Endpoints......Page 286
3.1 The ATBC and CARET Trials in Lung Cancer and the SELECT Trial in Prostate Cancer......Page 288
3.3 The BCPT and STAR Trial in Breast Cancer......Page 289
3.4 Trials of Nonsteroidal Anti-­inflammatory Drugs in Colorectal Cancer......Page 290
4 Conclusion......Page 292
References......Page 293
Index......Page 296