FLAVONOID INDUCTION OF CYTOCHROME P450 (CYP) IN HUMAN ESOPHAGEAL CARCINOMA CELLS: IMPLICATIONS FOR CHEMOTHERAPY By Ramaesela Sharon Molepo A thesis submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science September 2010 Supervisors: Dr Collet Dandara & Professor Rob Veale DECLARATION I declare that this thesis is my own, unaided work. It is being submitted for the Master of Science degree at the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination in any other university. _________________ (Signature of candidate) ________ Day of _____________ 2011 i ABSTRACT Esophageal cancer ranks as the second most common cancer among black males in South Africa and patients often seek medical attention when the cancer has already reached advanced stages. Thus, there is a lot of work investigating factors associated with increased risk, biomarkers for early diagnosis as well as a search for suitable drugs to treat esophageal cancer. In this project, flavonoids were used on esophageal cancer cell lines to identify enzymes that are differentially expressed as well as to evaluate the flavonoids? direct toxic effects on the cancer cells using ?-Naphthoflavone (BNF) on human esophageal cancer cell lines, WHCO1 and WHCO6. Differential expression of drug metabolising enzymes, CYP1A1, CYP1A2 and CYP1B1 was also investigated. BNF showed a moderate antiproliferative activity in WHCO6 cells (IC50~ 10?M) and a weak activity in WHCO1 cells (IC50~25 ?M). Thus, suggesting that esophageal cancer cells may be responsive to the treatment with BNF. BNF resulted in the differential expression of CYP1A1, CYP1A2, and CYP1B1. These results, implicate CYP1 enzymes as potential therapeutic targets for esophageal cancer prevention. ii ACKNOWLEDGMENTS I would like to show my deepest gratitude to my supervisors: Professor Rob Veale for his support, enthusiasm and profound knowledge of science. Dr Collet Dandara for believing in me and for coming up with such an interesting project. My sincere thanks also go to Mrs Elsabe Scott for her patience and for always providing me with cells. I also wish to thank members of the cell biology lab, Yael, Nicolene and Stephanie for their support and valuable advices, it has been a pleasure to work in a friendly and pleasant atmosphere. The generous financial support of the following organizations is gratefully acknowledged: University of the Witwatersrand and the Council for the scientific and industrial research (CSIR) Scholarship, University of the Witwatersrand and Bradlow bursary, The cancer association of south Africa (CANSA) (Grant to C Dandara) and the National research foundation (NRF) (Grant to R.B Veale). I am also heartily thankful to Mrs Kgadi Selepe for believing in me, her support and valuable advice on life. Last, but not least, I thank my family: My parents Merriam and Ramagodi Molepo for their unconditional support and encouragement to pursue my interests. My sister Molebogeng Molepo for listening to my complaints and frustrations, and for believing in me. iii LIST OF ABBREVIATIONS 2-OHE2 2-Hydroxyestradiol 4-OHE2 4-Hydroxyestradiol AFB1 Aflatoxin B1 AHR Aryl Hydrocarbon receptor AHRR Aryl hydrocarbon receptor repressor AIP Aryl Hydrocarbon Receptor interacting protein ANF ?-Naphthoflavone ANOVA One way analysis of variance ARNT Aryl hydrocarbon receptor nuclear translocator bHLH Basic helix-loop-helix BNF ?-Naphthoflavone BSA Bovine serum albumin BP Benzo(a)pyrene BPDE Benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide COMT Catechol O-Methyltransferase CYP Cytochrome P450 DMSO Dimethylsulfoxide E2 17-? Estradiol FBS Fetal bovine serum GST Glutathione-S-transferase HOSCC Human esophageal squamous carcinoma cell iv HRP Horseradish peroxidise conjugate HSP Heat shock proteins HSP90 Heat shock protein 90 IC50 Median inhibition concentration MTT 3-4, 5-Dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide PAHs Polyaromatic hydrocarbons S.D. Standard deviation SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SNPs Single nucleotide polymorphisms TBS Tris buffered saline TBST Tris buffered saline-Tween-20 TCA 7.5% tichloro-actic acid TCDD 2, 3, 7, 8-Tetrachlorodibenzo-P-dioxin XRE Xenobiotic response element v LIST OF FIGURES Figure 1.1 : A Simplified schematic showing the metabolism of xenobiotics.............................. 2 Figure 1.2 : Schematic representation of the human CYP1A1 gene .........................................6 Figure 1.3 : Metabolic activation of Benzo[a] pyrene into a carcinogenic Benzo(a)pyrene 7.8-diol-9,10-epoxide .....................................................................................7 Figure 1.4 : A schematic representation of the human CYP1A2 gene.......................................... 9 Figure 1.5 : Metabolic activation of Aflatoxin B1 to AF B1 ?8, 9-epoxide leading to the formation of AF B1 ?DNA adduct (AF B1 ?N7-guanine) ..............................11 Figure 1.6 : A schematic representation of the human CYP1B1 gene ....................................14 Figure 1.7 : Metabolic activation of estradiol and the formation of non-carcinogenic 2-methoxyestradiol and the carcinogenic 4-hydroxyestradiol .................. 17 Figure 1.8 : A schematic representation of the human AHR gene ..........................................20 Figure 1.9 : Outline of the function of aryl hydrocarbon receptor (AhR) as a Ligand-activated transcription factor ......................................................................................22 Figure 2.1 : The effect of DMSO on cell viability ..................................................................... 32 Figure 3.1 : Different types of flavonoid structures..............................................................37 Figure 3.2 : The effect of BNF on the proliferation of human esophageal carcinoma cells (WHCO1 and WHCO6) after 24 hours .......................................................43 Figure 3.3 : The effect of BNF on the proliferation of human esophageal carcinoma cells (WHCO1 and WHCO6) after 48 and 72 hours ............................................44 Figure 3.4 : The median inhibition concentration (IC50) of BNF in WHCO1 and WHCO6 cells .............................................................................................................................. 46 vi Figure 4.1 : SDS PAGE of samples extracted from WHCO1 and WHCO6 cell lines ...........57 Figure 4.2 : The effect of BNF on the expression of CYP1A1 protein in WHCO1 and WHCO6 cells ............................................................................................................................... 59 Figure 4.3 : The effect of BNF on the expression of CYP1A2 protein in WHCO1 and WHCO6 cells .........................................................................................................................60 Figure 4.4 : The effect of BNF on the expression of CYP1B1 protein in WHCO1 and WHCO6 cells ..........................................................................................................................61 Figure 4.5 : The effect of BNF on the expression of AHR protein in WHCO1 and WHCO6 cells ..................................................................................................... 62 Figure 7.1 : BSA standard curve for protein estimation ........................................................127 vii LIST OF TABLES Table 1.1.A : Human CYP genes and their functions ................................................................4 Table 4.1: Comparison of CYP1A1, CYP1A2 and CYP1B1 expression in BNF treated cells ..............................................................................................63 Table 1.1.B : One-Way ANOVA column statistics for DMSO treated WHCO1 cells ........106 Table 1.2 : Repeated measures ANOVA for DMSO treated WHCO1 cells .........................107 Table 1.3 : General ANOVA for DMSO treated WHCO1 cells............................................107 Table 1.4 : Tukey?s multiple comparison test showing the significant difference between the different DMSO concentrations used to treat WHCO1 cells ................................................108 Table 1.5 : One-way Anova column statistics for DMSO treated WHCO6 cells .................109 Table 1.6 : Repeated measures ANOVA for DMSO treated WHCO6 cells .........................109 Table 1.7: General ANOVA for DMSO treated WHCO6 cells ............................................... 110 Table 1.8 : Tukey?s multiple comparison test showing the significant difference between the different DMSO concentrations used to treat WHCO6 cells ................................................110 Table 1.9 : One-way ANOVA column statistics for WHCO1 cells treated with BNF for 24 hours ........................................................................................................................................... 111 Table 1.10 : Repeated measures ANOVA for WHCO1 cells treated with BNF for 24 hours ..........................................................................................................112 Table 1.11 : General ANOVA for WHCO1 cells treated with BNF for 24 hours ...............112 Table 1.12 : Tukey?s multiple comparison test showing the significant difference between the different BNF concentrations used to treat WHCO1 cells after 24 hours ............................113 viii Table 1.13 : One-way ANOVA column statistics for WHCO1 cells treated with BNF for 48 hours ......................................................................................................................................114 Table 1.14 : Repeated measures ANOVA statistics for WHCO1 cells treated with BNF for 48 hours ..........................................................................................................114 Table 1.15: General ANOVA statistics for WHCO1 cells treated with BNF for 48 hours .... 115 Table 1.16 : Tukey?s multiple comparison test showing the significant difference between the different BNF concentrations used to treat WHCO1 cells after 48 hours ............................115 Table 1.17: One-way ANOVA column st atistics for WHCO1 cells treated with BNF for 72 hours ........................................................................................................................................... 116 Table 1.18: Repeated measures ANOVA for WHCO1 cells treated with BNF for 72 hours ............................................................................................................... 116 Table 1.19: General ANOVA for WHCO1 cells tr eated with BNF for 72 hours ................... 117 Table 1.20 : Tukey?s multiple comparison test showing the significant difference between the different BNF concentrations used treat WHCO1 cells for 72 hours ...................................117 Table 1.21: One-way ANOVA column st atistics for WHCO6 cells treated with BNF for 24 hours ........................................................................................................................................... 118 Table 1.22 : Repeated measures ANOVA for WHCO6 cells treated with BNF for 24 hours ................................................................................................... 119 Table 1.23: General ANOVA for WHCO6 cells tr eated with BNF for 24 hours ................... 119 Table 1.24 : Tukey?s multiple comparison test showing the significant difference between the different BNF concentrations used to treat WHCO6 cells for 24 hours ...............................120 Table 1.25: One-way ANOVA column st atistics for WHCO6 cells treated with BNF for 48 hours ........................................................................................................................................... 121 ix Table 1.26: Repeated measures ANOVA for WHCO6 cells treated with BNF for 48 hours ............................................................................................................... 121 Table 1.27 : General ANOVA for WHCO6 cells treated with BNF for 48 hours ................122 Table 1.28: Tukey?s multiple co mparison test showing the significant difference between the different BNF concentrations used to treat WHCO6 cells for 48 hours ................................... 122 Table 1.29: One-way ANOVA column st atistics for WHCO6 cells treated with BNF for 72 hours ........................................................................................................................................... 123 Table 1.30 : Repeated measures ANOVA for WHCO6 cells treated with BNF for 72 hours ..........................................................................................................123 Table 1.31: General ANOVA for WHCO6 cells tr eated with BNF for 72 hours ................... 124 Table 1.32: Tukey?s multiple co mparison test showing the significant difference between the different BNF concentrations used to treat WHCO6 cells for 72 hours ................................... 124 Table 1.33 : The median inhibition concentration (IC50) values of BNF in WHCO1 cells ..125 Table 1.34: The median inhibition concentration (IC 50) values of BNF in WHCO6 cells ..... 125 Table 1.35: CYP1A1 fold induction ......................................................................................... 130 Table 1.36 : CYP1A2 fold induction ....................................................................................130 Table 1.37: CYP1B1 fold induction .......................................................................................... 131 Table 1.38 : AHR fold induction ...........................................................................................131 x Table of Contents Declaration.................................................................................................................... ..... . . . . . . . . i Abstract ........................................................................................................................................ii? Acknowle d g e m e n t s ....................................................................................................................iii? List of Abbreviations .......................................................................................................... ... . i v List of Figures .............................................................................................................................vi? List of Tables ............................................................................................................................ viii? Chapter 1: General introduc t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 . 1 . Variatio n in xenobiot i c metaboli s m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2. Cytochrome P450 (CYP) enzymes ..................................................................................... 3 1.3. Human xenobiot i c - metaboli z i n g CYP1 enz y me s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.3.1. CYP1A1 ....................................................................................................................... 5 1.3.1.1. Gene structure ........................................................................................................ 5 1.3.1.2. Metabolic reactions catalyzed by CYP1A1 ........................................................... 6 1.3.1.3. Tissue specific expression of CYP1A1.................................................................. 7 1.3.1.4. CYP1A1 Polymorphisms ....................................................................................... 8 1.3.2. CYP1A2 ....................................................................................................................... 9 1.3.2.1. Gene structure ........................................................................................................ 9 1.3.2.2. Metabolic reactions catalyzed by CYP1A2 ......................................................... 10 1.3.2.3. Tissue specific expression of CYP1A2................................................................ 12 1.3.2.4. CYP1A2 Polymorphisms ..................................................................................... 12 1.3.3. CYP1B1 ..................................................................................................................... 13 1.3.3.1. Gene structure ...................................................................................................... 13 1.3.3.2. Metabolic reactions catalyzed by CYP1B1 .......................................................... 14 1.3.3.3. Tissue specific expression of CYP1B1 ............................................................... 17 1.3.3.4. CYP1B1 Polymorp h i s ms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8 1.3.4. The Aryl hydrocar b o n receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9 1.3.4.1. Gene structure ...................................................................................................... 19 1.3.4.2. The AHR pathway ................................................................................................ 20 1.3.4.3. Regulati o n of the AHR pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 1.3.4.4. Association of AHR polymo r p h i s ms with human diseas e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 4 1.4. Cancer Che motherapy ....................................................................................................... 25 xi 1.5. AIMS................................................................................................................................. 26 Chapter 2: The model ............................................................................................................ 27 2 . 1 . Introduc t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 27 2.2. Materials and Methods ...................................................................................................... 29 2.2.1. Cell line and Cell Culture ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 9 2.2.2. Cell treatme n t ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 29 2.2.3. MTT Cell Viability Assay ......................................................................................... 30 2.2.4. Statistical Analysis .................................................................................................... 30 2.3. Results .............................................................................................................................. 31 2.4. Discussi o n and Conclusi o n ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 Chapter 3:The value of BNF as an anti proliferative agent for esophageal cancer cells ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... 36 3 . 1 . Introduc t i o n ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 36 3.2. Materials and Methods ..................................................................................................... 39 3.2.1. Cell line and Cell Culture ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9 3.2.2. Cell treatme n t ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 39 3.2.3. MTT Cell Viability Assay ......................................................................................... 40 3.2.4. Determi n i n g the median i nhibi t i o n concen t r a t i o n (IC 50 ) ........................................... 40 3.2.5. Statistical Analysis .................................................................................................... 40 3.3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ 42 3.3.1. The antiprol i f e r a t i v e activity of BNF against WHCO1 and WHCO6 esophageal cancer cells ....................................................................................................... 42 3.3.2. The median inhibition concentra tion (IC50) values of BNF in WHCO1 and WHCO6 c ells ................................................................................................. 45 3.4. Discussi o n and Conclusi o n ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 7 Chapter 4: The effects of BNF on the expression of CYP1A1, CYP1A2, CYP1B1 and AHR in the two esopha g e a l can cer cell lines, WHCO1 and WHCO6 ....... . . . . . . . . . . . . . . . . . . . . . . . 50 4 . 1 . Introduc t i o n ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 50 4.2. Materials and Methods ..................................................................................................... 52 4.2.1. Cell Culture and Treatme n t ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 4.2.2. Whole Cell protein Extracti o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 4.2.3. Protein Estimati o n ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 4.2.4. SDS-PAGE ............................................................................................................... . 54 4.2.5. Western Blotting ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 xii 4 . 2 . 6 . Densitome t r y ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 55 4.2.7. Statistical Analysis .................................................................................................... 55 4.3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ 56 4.3.1. SDS-PAGE for protein estimati o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 6 4.3.2. The effect of BNF on CYP1 A1, CYP1A2, CYP1B1 and AHR protein expression ...................................................................................................... 57 4.3.2.1. The effect of BNF on CYP1A1 protein express i o n ....... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 8 4.3.2.2. The effect of BNF on CYP1A2 protein expression ............................................ 59 4.3.2.3. The effect of BNF on CYP1B1 protein expression ............................................ 60 4.3.2.4. The effect of BNF on AHR protein expressi o n ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2 4.3.3. BNF induces the expression of CYP1B1 protein to significan t l y higher levels than CYP1A1 and CYP1A2 protei n s in WHCO1 and WHCO6 cells .......................................................................................................................... 63 4.4. Discussion ........................................................................................................................ 64 4.4.1. BNF induces the expressi o n of CYP1 enzyme s ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4 4.4.2. BNF down-regulates the expression of AHR ............................................................ 67 4.5. Conclusion ....................................................................................................................... 69 Chapter 5: General discussi o n ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 References ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... 77 Appendices ........................................................................................................................... 105 Appendix 1 ............................................................................................................................ 105 1.1. MTT cell viability assay .............................................................................................. 105 Appendix 2 ............................................................................................................................ 106 2.1. The effe ct of DMSO on WHCO1 cell viabilit y (One-Wa y anova and Tukey posttest ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 6 Appendix 3 ............................................................................................................................ 109 3.1. The effe ct of DMSO on WHCO6 cell viabilit y (One-Wa y anova and Tukey posttest ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 9 Appendix 4 ............................................................................................................................ 111 4.1. The effe ct of BNF on WHCO1 cell prolifer a t i o n (One-way anova and Tukey posttest) ..................................................... 111 Appendix 5 ............................................................................................................................ 118 xiii 5.1. The effe ct of BNF on WHCO6 cell prolifer a t i o n (One-way anova and Tukey posttest) ..................................................... 118 Appendix 6 ............................................................................................................................ 125 6.1. The me dian inhibiti o n concentr a t i o n (IC50) values .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 5 Appendix 7 ............................................................................................................................ 126 7.1. Protein extracti o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 7.2. Protei n estimati o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 7.3. SDS-PAGE .................................................................................................................. 127 7.4. Western Blot ................................................................................................................ 128 Appendix 8 ............................................................................................................................ 130 8.1. T-test results for compari ng protein expression level ................................................. 130 ? ? ? ? xiv CHAPTER 1: General introduction 1.1. Variation in xenobiotic metabolism Humans are continuously exposed to harmful xenobiotic compounds which if allowed to accumulate in their bodies could become toxic. Most of these xenobiotics require to be changed or biotransformed in order to be removed from the body. The biotransformation results in mainly detoxification of the xenobiotics but in some cases, metabolic activation occurs. Detoxication or biotranformation of xenobiotics is catalysed by enzymes that belong to gene superfamilies. Detoxification or metabolism of xenobiotics is classified into three phases, I, II and III. The phase I enzymes most of which are the cytochrome P450 (CYP) enzymes, catalyze the functionalization reactions resulting in more polar metabolites (Conney, 2003; Guengerich, 2004; Caro and Cederbaum, 2004; Coon, 2005; Kim and Guengerich, 2005). The phase II enzymes, such as the glutathione transferases, UDP glucuronosyltransferases and N-acetyl transferases, conjugate the phase I products with water-soluble endogenous metabolites such as glutathione, glucuronic acid, sulfate, cysteine or acetate, producing hydrophilic products that can be easily excreted (Burchell, 2003; Kuuranne et al; 2003). However, the phase I products are not always conjugated by phase II enzymes since the phase I products are often highly reactive metabolites and can bind to macromolecules such as proteins and nucleic acids (Figure 1.1), thus leading to cancer development 1 (Guengerich, 2000, Schwarz et al, 2001). Phase III metabolism is involved in the transport of the conjugated xenobiotics. Individuals with an imbalance in phase I, phase II enzyme and phase III activity may have a differential susceptibility to cancer. This project focuses on the CYP enzymes, since these enzymes play a major role in the toxicity and carcinogenesis of several xenobiotic compounds. Figure 1.1 : A Simplified schematic showing the metabolism of xenobiotics. Xenobiotics entering the cell are metabolized by phase I enzymes and detoxified by phase II enzymes and their transport from the cells is facilitated by phase III enzymes. Phase I metabolites may either bind to macromolecules thus causing cancer or be conjugated with water-soluble endogenous molecules leading to excretion of these metabolites (Oyama et al, 2003). 2 1.2. Cytochrome P450 (CYP) enzymes Cytochrome P450 (CYP) enzymes are a superfamily of hemoprotein monooxygenases that catalyze the oxidation of a wide variety of both endogenous compounds such as steroids, bile acids and fatty acids, and xenobiotic compounds including drugs, toxins and carcinogens (Caro and Cederbaum, 2004). The CYP enzymes or microsomal carbon monoxide binding pigments as they were initially called, were first discovered in the late 1950?s when Klingenberg identified a pigment in the rat liver that produced a unique spectrum ranging between 350 and 500nm upon reduction with carbon monoxide and gave an optical absorption peak at 450nm (Klingenberg, 1958). A few years later, the hemoprotein nature of this pigment was identified and was then given the name cytochrome P450 (Omura and Sato, 1964). CYP enzymes have been identified in all studied organisms, from bacteria to humans (Nelson et al, 2004). Fifty seven active (57) genes encoding cytochrome P450 and fifty eight(58) pseudogenes have been identified in humans to date (Nelson et al, 2004, Nelson, 2009).Using sequence homology, the CYP enzymes have been grouped into families and subfamilies and there are eighteen(18) families and fourty three(43) subfamilies currently known in humans (Nelson et al, 2004). The majority of CYP enzymes metabolize endogenous substrates in a highly substrate-specific manner (Lang et al, 2001; 3 Jinno et al, 2003; Nebert and Dalton, 2006) while others show some overlap in substrate specificity (Yamazaki et al, 2001; Shimada and Fujii-Kuriyama, 2004; Mahadevan et al, 2007). Of the fiftyseven (57) CYP genes found in humans it is mostly the members of the CYP1, CYP2 and CYP3 families that are responsible for xenobiotic metabolism, while other CYP families only metabolize endogenous substrates (see Table 1.1.).The focus of this project will be on the CYP1 family of enzymes. Table 1.1.: Human CYP genes and their functions Family Members Main substr a t e s / p at h w ay CYP1 3 Subfamilies, 3 genes, 1 pseudogene Polyaromatic hydrocarbon CYP2 13 subfamilies, 16 genes,16 pseudogenes Caffeine and testosterone CYP3 1 subfamily,4 genes, 2 pseudogenes Xenobiotics, steroids CYP4 6 subfamilies, 11 genes, 10 pseudogenes Arachidonic acid CYP5 1 subfamily, 1 gene Thromboxane synthesis CYP7 2 subfamilies, 2 genes Cholesterol 7?-hydroxylation CYP8 2 subfamilies, 2 genes Prostacyclin synthesis CYP11 2 subfamilies, 3 genes Steroid biosynthesis CYP17 1 subfamily, 1 gene Steroid 17?-hydroxylation CYP19 1 subfamily, 1 gene Androgen aromatization CYP20 1 subfamily, 1 gene Unknown CYP21 2 subfamilies, 2 genes,1 pseudogene Steroid biosynthesis CYP24 1 subfamily, 1 gene Vitamin D degradation CYP26 3 Subfamilies, 3 genes Retinoic acid hydroxylation CYP27 3 subfamilies, 3 genes Steroid 27-hydroxylation CYP39 1 subfamily, 1 gene Cholesterol CYP46 1 subfamily, 1 gene Cholesterol 24-hydroxylation CYP51 1 subfamily, 1 gene, 3 pseudogene Cholesterol biosynthesis Data extracted from Lund et al, 1999 and Nelson, 1999 4 1.3. Human xenobiotic- metabolizing CYP1 enzymes The members of the CYP1 family are, CYP1A1, CYP1A2, CYP1B1 and a pseudogene (Nelson, 2009). The CYP1 enzymes have been studied extensively because of the major roles they play in chemical carcinogenesis. They catalyze the bioactivation of mostly polyaromatic hydrocarbons such as benz[a]anthracene, 7,12- dimethylbenz[a]anthracene, arylamines, and benzo(a)pyrene (BP) (Murray et al, 2001; Iwanari et al , 2002 ; Shimada and Fuji-kuriyama, 2004). The same substrates are also known to act as their inducers (Iwanari et al 2002; Guruge et al, 2009). The three CYP1 genes are expressed in a tissue specific manner (Jana et al, 2000; Murray et al, 2001; Coumoul et al, 2001; Iwanari et al, 2002;) and their transcriptional regulation is via the aryl hydrocarbon receptor complex (Nebert et al, 2004). The functions, tissue-specific expression and the regulation of these genes are discussed in the following sections. 1.3.1. CYP1A1 1.3.1.1. Gene structure The human CYP1A1 gene is located on chromosome 15 on loci 15q22-qter and is composed of seven exons and six introns (Jaiswal et al, 1987; Corchero et al, 2001). The CYP1A1 gene codes for a 2.8 kb mRNA and a protein of 512 amino acids. Figure 1.2 shows a schematic of the gene organisation for CYP1A1. 5 Figure 1.2 .: Schematic representation of the human CYP1A1 gene (adapted from Jaiswal et al, 1987 & Corchero et al, 2001). 1.3.1.2. Metabolic reaction s catalyzed by CYP1A1 Human CYP1A1 is one of the key enzymes in the activation of pro- carcinogenic polycyclic aromatic hydrocarbons (PAHs) (Guengerich, 1992; Shimada et al, 1992; Guengerich et al, 1999; Minsavage et al, 2004; Schwartz et al, 2007). PAHs are the major carcinogens found in the environment as pollutants. Prolonged exposure to PAH contaminated air has been associated with increased lung cancer risk (Boffetta et al, 1997). A classic example is the activation of benzo (a) pyrene (BP). BP is a PAH generated during incomplete combustion such as in cigarette smoke (Pfeifer et al, 2002). BP has been associated with immune suppression, birth defects and carcinogenesis (Miller and Ramos, 2001). CYP1A1 biotransforms BP to produce the ultimate carcinogen BP-7, 8-dihydrodiol-9, 10-epoxide (BPDE). BPDE can covalently bind to DNA thus causing guanine (G) to thymine (T) transversions (Tokiwa et al, 1993; Conney et al, 1994; Nebert et al, 2000; Schwarz et al, 2001; Pfeifer et al, 2002;). These transversions have b een found to activate a number of transcriptional factors such as P53 (Blattner et al, 1999; Ueno et al, 1999). An 6 excessive amount of G to T transversions in the P53 gene has been associated with lung cancer. Figure 1.3 is a diagrammatic representation of a reaction catalysed by CYP1A1 and epoxide hydrolase (EH) resulting in the formation of the reactive metabolite, BPDE that covalently binds to DNA resulting in DNA damage and ultimately cancer. Figure 1.3 : Metabolic activation of Benzo[a] pyrene into a carcinogenic Benzo (a) pyrene 7.8-diol-9, 10-epoxide. (Adapted from Kim et al, 1998) 1.3.1.3. Tissue specific expression of CYP1A1 CYP1A1 constitutive expression is very low in extrahepatic tissues (Shimada et al, 1992). However, high levels of this enzyme have been reported in almost every tissue studied, including vascular endothelium, smooth muscle cells, lung gastrointestinal tract, placenta, brain and others (Celander et al, 1997: Zhao et al, 1998; Ding and Kaminsky, 2003), only in the presence of an inducer. CYP1A1 is highly inducible by PAHs such as 2, 3, 7, 8-tetrachloro- dibenzo-p-dioxin (TCDD) (Whitlock, 1999; Kim and Sh een, 2000; Chang et al, 2009), BP, 3-methylcholanthrene (3MC) and omeprazole (Whitlock, 1999; 7 Bowen et al, 2000; Rodriguez-Antona et al, 2000), through the aryl hydrocarbon receptor pathway (Whitlock, 1999; Denison and Nagy, 2003). Previous studies have shown CYP1A1 to be highly expressed in tumors compared with the surrounding normal tissues. For example, CYP1A1 was shown to be over expressed in ovarian cancer cells and esophageal tumors as opposed to normal cells (Murray et al, 1994; Nakajima et al, 1996; Leung et al, 2005). Recently, CYP1A1 was found to be highly expressed in oral squamous cell carcinoma after BP treatment (Hecht, 2003; Nagaraj et al, 2006; Chi et al, 2009). Therefore, CYP1A1 can be suggested as a target for novel chemotherapeutic agents (Mcfadyen et al, 2004; Rooney et al, 2004). 1.3.1.4. CYP1A1 Polymorphisms Several CYP1A1 single nucleotide polymorphisms (SNPs) have been reported in the human population (http:// www.cypalleles.ki.se/). Four common SNPS in CYP1A1 have been studied concerning their potential implications in the risk of breast cancer. These SNPs include T3801C, T3205C, A2455G and C2453A (Ambrosone et al, 1995; Taioli et al, 1995; Huang et al, 1999). CYP1A1 SNPs have also been reported to be associated with other cancer types such as prostate (Acevedo et al, 2003; Chang et al, 2003, Suzuki et al, 2003; Aktas et al, 2004; Quinones et al, 2006, Shaik et al, 2009), colorectal (Jin et al, 2011) esophageal (Zhuo et al , 2009), oral and pharyngeal (Varela-Lema et al, 2008), lung cancer (Shi et al, 2008, San Jose et al, 2010), ovarian, endometrial and 8 cervical cancer (Murata et al, 1998; Longuemaux et al, 1999; Esteller et al, 1999; Goodman et al, 2001a; 2001b). 1.3.2. CYP1A2 1.3.2.1. Gene structure The human CYP1A2 loci is found on chromosome 15q22-qter and is composed of seven exons, six introns (Corchero et al, 2001), a 3.2 kb mRNA and a protein of 515 amino acids as shown in Figure 1.4 below. The CYP1A1 and CYP1A2 genes are separated by 23 kb and are orientated in opposite directions (Corchero et al, 2001). There is no open reading frame between these two genes, thus showing that they share a 5?-flanking region and share 80% amino acid sequence homology. Figure 1.4 : A schematic representation of the human CYP1A2 gene (adapted from Corchero et al, 2001). 9 1.3.2.2. Metabolic reaction s catalyzed by CYP1A2 CYP1A2 is responsible for the oxidative metabolism of a wide range of therapeutic drugs such as theophylline, lidocaine, imipramine, verapamil and phenothrazines (Yamazaki et al, 2001; Daniel et al , 2002; Llibre et al , 2002). This enzyme is also involved in the activation of endobiotic compounds such as melatonin, estradiol and uroporphy rinogen (Nichols et al, 2003; Tsuchiya et al, 2005; Faber et al, 2005; Ma and Lu, 2007). In addition, CYP1A2 metabolizes various carcinogenic compounds, such as heterocyclic and aromatic amines to reactive metabolites, leading to toxicity and cancer (Brosen, 1995; Guengerich et al , 1999; Van schaik, 20 05; Ma and Lu, 2007). Aflatoxin B1 (AFB1) is a potent food carcinogen and has been reported as a causative agent in hepatocellular carcinoma (Kew, 2003; Sudakin, 2003; Williams et al, 2004). AFB1 on its own is not a potent toxin since it requires metabolic activation to exert its genotoxicity. CYP1A2 catalyzes the biotransformation of AFB1 to the highly reactive AFB1-8, 9-epoxide (McLean and Dutton, 1995; Guengerich et al, 1998) (Figure 1.5.). This epoxide is a mixture of both the exo- and endo-8, 9-epoxide (Ueng et al, 1995). Only the exo-8, 9-epoxide has been reported to have genotoxic characteristics (Guengerich et al, 1998). Exo-8, 9-epoxide is highly unstable and binds to guanine bases in DNA thus forming aflatoxin-N7-guanine (Guengerich, 2001; Nayak et al, 2001). The aflatoxin-N7-guanine has been reported to be capable 10 of forming guanine to thymine transversion mutations in DNA (Bailey et al, 1996) (Figure 1.5). Figure 1.5 : Metabolic activation of Aflatoxin B1 to AFB1 ?8, 9-epoxide leading to the formation of AFB ?DNA adduct (AFB ?N71 1 -guanine) (Chou and Chen, 1997). 11 1.3.2.3. Tissue specific expression of CYP1A2 Human CYP1A2 is constitutively expressed in the liver (Mckinnon et al, 1991; Schweikl et al, 1993; Landi et al, 1999), thus accounting for 13% of the total CYP in this organ. However, a few studies have reported the expression of this gene in other organs such as the lung (Mac? et al , 1998; Wei et al, 2001; Bernauer et al, 2006), esophagus (Lechevrel et al, 1999), brain (Bhagwat et al, 2000), prostate (Williams et al, 2000) and gastrointestinal tract (Tatemichi et al, 1999). The constitutive level of expression of the CYP1A2 gene is increased by exposure to PAHs from cigarette smoke and TCDD via the Aryl hydrocarbon receptor/Aryl nuclear translocator (AHR/ARNT) complex (Shen et al, 1994; Hankinson et al, 1995; Landi et al, 1999). 1.3.2.4. CYP1A2 polymorphisms Several CYP1A2 polymorphisms have been reported in the past few years (http://www.cypalleles.ki.se). These pol ymorphisms are associated with individual differences in CYP1A2 activity (Saruwatari et al, 2002; Zhang et al, 2007). It has been demonstrated that the CYP1A2 SNP G3860A is associated with decreased enzyme inducibility in Japanese smokers (Nakajima et al, 1999). The CYP1A2 SNP C163A was also reported to be associated with higher enzyme inducibility in white smokers (Sachse et al, 1999). In a recent study the CYP1A2 haplotype (T739G, C729T, and C163A) was reported to be linked with decreased enzyme activity in Ethiopian non-smokers (Aklillu et al, 12 2003). These interindividual differences influence individual susceptibility to cancer risk caused by procarcinogens and the therapeutic efficacy of drugs. Recently, low inducibility of CY1A2 polymorphism was reported to be associated with a high risk of myocardial infarction (Cornelis et al, 2004). 1.3.3. CYP1B1 1.3.3.1. Gene structure The human CYP1 gene family was initially thought to consist of CYP1A1 and CYP1A2 only. It was only in 1994 that a new member of the CYP1 gene family, CYP1B1, was discovered. This gene was induced in human keratinocyte cell line treated with tetrachloro-dibenzo-p-dioxin (Sutter et al, 1994). The CYP1B1 cDNA was cloned to facilitate the characterization of the protein. Nucleic acid and amino acid sequence analysis revealed ~40% homology of CYP1B1 with CYP1A1 and CYP1A2. There are various distinct properties that separate CYP1B1 from CYP1A1 and CYP1A2. CYP1B1 gene is located on position 2p21-22, spanning approximately 12 kilobases (kb) of DNA and contains three exons and two introns (Tang et al, 1996). Although it has the simplest gene structure, CYP1B1 is one of the largest known human CYPs in terms of the mRNA size and number of amino acids. CYP1B1 has a 5.2kb mRNA with an open reading frame beginning at the 5? end of the second exon (Murray et al, 2001). The predicted protein sequence of this CYP is 543 amino acids (Figure 1.6) and this differs with other CYPs which have their 13 open reading frames beginning in exon 1 (Murray et al, 2001). Figure 1.6 below shows the schematic organisation of the CYP1B1 gene. Figure 1.6: A schematic representation of the human CYP1B1 gene (adapted from Murray et al, 2001). 1.3.3.2. Metabolic reaction s catalyzed by CYP1B1 The human CYP1B1 activates many structurally diverse environmental procarcinogens, such as PAHs, heterocyclic and aryl amines, as well as the nitroaromatic hydrocarbons (Shimada et al, 1996; Kim et al, 1998; Guengerich, 2000; Guengerich et al, 2003). CYP1B1 also detoxifies cancer drugs such as flutamide and docetaxel, and this reduces their cytotoxic potential and effectiveness (Rochat et al, 2001; MacFadyen et al, 2001; Bournique and Lemarie, 2002). CYP1B1 has also been found to be effective in activating aflatoxin B1 to its mutagenic metabolite (Crespi et al, 1997). When expressed in S.cerevisiae, the human CYP1B1 was found to metabolize a dietary heterocyclic amine, 2-amino-1 methyl-6-phenylimidazo [4, 5-b] 14 pyridine which has been associated with breast and colon cancer (Crofts et al, 1997). In addition to its role in procarcinogen activation, CYP1B1 is also a key enzyme involved in the production of potentially carcinogenic estrogen metabolites. Estrogens have various biological effects, such as female sexual differentiation and development, and arterial vasodilation, the maintenance of bone density and neuroprotective actions. However, prolonged exposure to estrogens has been associated with breast cancer and endometrial cancer (Pike et al, 1993; Jordan, 2000; and Liehr, 20 00). Xenoestrogens, such as oral contraceptives and drugs for hormone replacement therapy are also associated with breast cancer risk through their estrogenic properties 17-? estradiol (E2) is the most common steroidal estrogen present in women. The principal source of this estrogen is from pre-menopausal females, ovary and adrenal glands .CYP1B1 encodes for an enzyme that metabolizes E2 to catechol estrogens 4-hydroxyestradiol (4-OHE2) and 2-Hydroxy estradiol (2- OHE2) (Figure 1.7). This enzyme usually produces higher levels of 4-OHE2 than 2-OHE2 (Yager and Liehr, 1996; Hayes et al , 1996; Spink et al, 1997; Cavalieri et al, 1997; Badawi et al, 2001; Cavalieri and Rogan, 2004). The 4- OHE2 gets oxidized to E2-3, 4-Quinone which readily reacts with DNA thus 15 causing damage to the DNA (Nutter et al, 1991; Nutter et al, 1994; Cavalieri et al, 1997) (Figure 1.7). High levels of 4-OHE2 have been detected in human uterine myometrium, benign uterine leiomyoma (Liehr et al, 1995), as well as in benign and malignant mammary tumors (Lemon et al, 1992; Liehr and Ricci, 1996). Human breast cancer tissues have been shown to have significantly higher levels of 4-OHE2 than normal breast tissues (McKay et al, 1995; Liehr and Ricci, 1996; Murray et al, 1997).The catechol estrogen 4- OHE2 has also been shown to be carcinogenic in animal models (Kristensen and Borresen-Dale, 2000; Hanna et al, 2000). CYP1B1 is therefore one of the risk factors for breast cancer because of the role it plays in steroid metabolism. 16 Figure 1.7 : Metabolic activation of estradiol and the formation of non- carcinogenic 2-methoxyestradiol and the carcinogenic 4-hydroxyestradiol. Abbreviations used CYP1B1 (Cytochrome P450 1B1), COMT (Catechol O- methyltransferase) (adapted from Chun and Kim, 2003). 1.3.3.3. Tissue specific expression of CYP1B1 The human CYP1B1 mRNA and protein are constitutively expressed in extrahepatic tissues such as the ovaries, testes, adrenal glands, breast, uterus and prostate, but are poorly expressed in the liver, kidney and lung tissues (Shimada et al, 1996; Hakkola et al , 1997 and Tang et al, 1999). One of the most significant discoveries regarding CYP1B1 expression is its high frequency of expression in various types of tumors compared to the corresponding normal tissues (Murray et al, 1997; Cheung et al, 1999; 17 McFadyen et al, 1999). For example, the expression of CYP1B1 was detected in breast tumors, but not in normal breast tissues (Huang et al, 1996; Kaminsky and Spivack, 1999). In one study, CYP1B1 was found to be over expressed in prostate tumors when compared to the normal prostate tissues (Finnstr? m et al, 2001; Chaib et al , 2001; Carnell et al , 2004). Together these findings implicate CYP1B1 as a potential therapeutic target for reducing the development of a wide variety of cancers and that this CYP may play a major role in anticancer drug resistance (McFadyen et al, 2001; Rochat et al, 2001). 1.3.3.3. CYP1B1 polymorphisms Six different polymorphisms of the CYP1B1 gene have been reported and four of these result in amino acid substitutions (Bailey et al, 1998; Stoilov et al, 1998; Mitrunen and Hirvonen, 2003). Tw o of these amino acid substitutions are encoded by exon 3, which encodes the heme-binding domain, at codon 453 Asn is substituted with Ser (Asn453Ser) (Bailey et al, 1998). The other amino acid substitutions are encoded by exon 2 (Stoilov et al, 1998), and they include, Arg48Gly and Ala119Ser. The CYP1B1 polymorphisms have been reported to have an association with breast or endometrial cancer risk (Bailey et al, 1998; Zheng et al, 2000; De vivo et al, 2002; Kocabas et al, 2002; Lee et al, 2003 and Sasaki et al, 2003; 18 Ozbek et al, 2010). These polymorphisms have also been found to lead to alterations in estrogen metabolism, thus causing 2 to 3 fold higher catalytic activity than the wild type enzyme (Shimada et al, 1999 and Hanna et al, 2000). For example, the Leu432Val polymorphism was reported to have the highest effect on the catalytic properties of the enzyme since the Val432 variant displayed three-fold higher 4-hydroxylase activity than the Leu432 variant (Li et al, 2000; Hanna et al, 2000 and Aklillu et al, 2002). Other studies also showed that Val432 variant is associated with higher 4-hydroxyestradiol: 2-hydroxyestradiol and 4-hydroxyestrone: 2-hydroxyestrone ratios than the Leu432 variant (Shimada et al, 1999). Higher catalytic activities towards estrogen metabolism have also been reported for the Gly48, Ser119, and Ser453 polymorphisms (Hanna et al, 2000). 1.3.4. The Aryl hydrocarbon receptor 1.3.4.1. Gene structure The Aryl hydrocarbon receptor (AHR) is a ligand activated basic helix-loop- helix (bHLH) protein that belongs to the Per-Arnt-Sim (PAS) family of transcription factors (Schmidt and Bradfield, 1996). It regulates the expression of human CYP genes (Quattrochi et al, 1994; Tang et al, 1996; Whitlock, 1999; Nebert et al, 2000) as well as some Phase II metabolizing enzymes (Hankinson, 1995; Schimidt and Bradfi eld, 1996; Hahn, 200 2). The AHR was first discovered in the livers of the C5BL/6 mice treated with TCDD (Bradfield 19 et al, 1991). In humans, this receptor encompasses 47,146 nucleotides and is located on chromosome 7 at location 7p15-21 (Micka et al, 1997). The human AHR consists of 11 introns and 12 exons, which encode an mRNA of 5.483 nucleotides thus giving rise to a protein with 848 amino acids (Harper et al, 1991). An illustration on the structure of how the gene is organised is given in Figure 1.8 below. Figure 1.8: A schematic representation of the human AHR gene (adapted from Harper et al, 1991). 1.3.4.2. The AHR pathway In its inactive form, the AHR is localized in the cytoplasm as a complex with a dimmer of heat shock proteins (HSP90), and one molecule of the AHR interacting protein (AIP) (Perdew, 1988; Holmes and Pollenz, 1997; Petrulis and Perdew, 2002; Hollingshead et al, 2004; Bock and Kohle, 2006), which are responsible for the AHR folding and stabilization (Gu et al, 2000). Halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons such as polychlorinated biphynyls, BP and polychlorinated dibenzo-p-dioxins 20 are the most common classes of the AHR ligands known (Denison et al, 1998; Denison and Heath-pagliuso, 1998). AHR ligands enter the cell by passive diffusion, where they encounter and bind to the AHR. Upon ligand binding, the AHR dissociates from the associated proteins and form a ligand-AHR complex. This complex translocates into the nucleus where it forms a heterodimer with the aryl nuclear translocator (ARNT) (McGuire et al, 1994; Goldberg, 1997; Heid et al, 2000; Fujii-Kuriyama and Mimura, 2005). The liganded AHR-ARNT heterodimer binds to specific regions of DNA known as xenobiotic response elements (XRE) thus regulating the expression of numerous genes, including CYP1A1, CYP1A2, and CYP1B1, in a positive or negative way as shown in Figure 1.9 (Hankinson, 1995; Nebert et al, 2000; Mimura and Fujii-Kuriyama, 2003; Okey et al, 2005; Ramadoss et al, 2005). 21 C ytoplasm Figure 1.9 : Outline of the function of aryl hydrocarbon receptor (AHR) as a ligand-activated transcription factor. AHR, Aryl hydrocarbon receptor; ARNT, AH- receptor-nuclear-translocator; CYP 1, Cytochrome P450 1; H SP90, heat shock protein 90 (shown in yellow), AIP, AHR interacting protein (shown in black); L, Ligand; XRE, Xenobiotic responsive element (adapted from Pollenz, 2002) 1.3.4.3. Regulation of the AHR pathway The AHR signalling may be down-regulated by several mechanisms; one of which is through the degradation of AHR protein through the ubiquitin- proteasome pathway (Pollenz, 2002). This pathway involves the binding of ubiquitin to the target protein (Pahl and Baeuerle, 1996; Tanaka, 1998, L A H R XR E C YP1 L A R N T A H R A R N T L Nucleus Ubiquitination Degradation L metabolism 22 Ciechanover et al, 2000; Kornitzer an d Ciechanover, 2000). The ubiquitinated protein is then degraded rapidly by the 26S proteasome (Pahl and Baeuerle, 1996; Tanaka, 1998, Ciechanover et al, 2000; Kornitzer and Ciechanover, 2000). The ubiquitin proteasome degradation pathway plays a role in the regulation of the AHR signal transduction pathway (Pollenz, 1996; Pollenz et al, 1998; Giannone et al, 1998; Pollenz and Barbadour, 2000; Ma and Baldwin, 2000). The degradation of AHR through the ubiquitin proteasome pathway can occur either in the cytosol or nucleus (Pollenz, 2002). For AHR degradation to occur the AHR/ARNT complex dissociates from the XRE and the ARNT, ubiquinated in the nucleus and gets degraded (Pollenz et al, 1994; Pollenz, 1996; Pollez, 2002) (Figure 1.9). Alternatively, after dissociating from the XRE and ARNT, AHR can be exported from the nucleus, ubiquinated in the cytoplasm and gets degraded by the 26S proteasome (Pahl and Baeuerle, 1996; Tanaka, 1998; Davarinos and Pollenz, 1999; Ciechanover et al, 2000; Kornitzer and Ciechanover, 2000; Pollenz, 2002). The other mechanism responsible for AHR downregulation involves the aryl hydrocarbon receptor repressor (AHRR) which acts as a negative regulator of the AHR function by competing with AHR for ARNT (Gradin et al, 1993; 23 Mimura et al, 1999; Gradin et al, 1999). A liganded AHR in a heterodimer with ARNT activates the expression of the AHRR gene (Mimura et al, 1999). The expressed AHRR inhibits the activity and binds to the ARNT, thus forming the AHRR-ARNT heterodimer (Gradin et al, 1993; Mimura et al, 1999; Haarmann-Stemmann and Abel, 2006) . This heterodimer binds to the XREs in DNA. 1.3.4.4. Association of AHR polymo rphisms with human diseases Human genetic variation has been widely studied for phase I and phase II metabolizing enzymes (Nebert et al, 1999; Park et al, 2000; Kalow, 2001; Ingelman-Sundberg, 2001; Lin and Lu, 2001; Miller and Kumar, 2001; Xie et al, 2001). Most of the sequence variants that have been reported in human AHR protein are due to SNPs. An association of SNPs with complex diseases such as cancer has been reported in most human genes; however, no striking effects of AHR SNPs on human health have been reported. For example, human exposure to dioxin-like compounds such as TCDD has been shown to cause chloracne, which is a severe skin disorder (Guo et al, 1999; Geusau et al, 2001). However, in one study, no association between the codon 554 polymorphism and chloracne was found in Caucasian population (Wanner et al, 1999). To date, no study has 24 reported a link between cancer and AHR polymorphism. For example, in previous studies there was no link found between the polymorphism associated with position 554 and lung cancer risk in Japanese smokers (Kawajiri et al, 1995). Cauchi et al, (2001), further confirmed this when he reported no link between the G459A polymorphism and lung cancer risk. Recently, Zhang et al, (2002) also found no association of the codon 554 polymorphism with bladder cancer risk in Chinese population. 1.4. Cancer Chemotherapy Each year an estimated 11 million people are diagnosed with cancer worldwide (Ferlay et al, 2004). Current chemotherapeutic options for cancer include drugs most of which have been shown to possess severe side effects (Marsh and McLeod, 2007). Due to this, there is a focus towards new chemotherapeutic agents that show limited toxicity to normal tissue as well as limited side effects. Flavonoids represent a large class of phenolic compounds present in plants. These compounds have often been associated with cancer prevention (Messina et al, 2006; Verheus et al, 2007; Arroo et al, 2008). In recent studies, their interaction with CYPs has been of particular interest. Flavonoids have been shown as inhibitors of CYP1A and CYP1B1 enzyme activity (Kim et al, 2005; 25 Chang et al, 2006) thus blocking the activation of procarcinogens into crcinogens (Shimada and Kuriyama, 2004). In the current study, the effect of ?-Naphthoflavone (BNF), a synthetic derivative of a naturally occurring flavonoid, on CYP1 expression was investigated. This is the first study that reports the effect of BNF on the expression of CYP1 enzymes in esophageal cancer cells WHCO1 and WHCO6 cells. 1.5. AIMS 1. To evaluate the antiproliferative activity of a synthetic flavonoid, ?- Naphthoflavone, on esophageal cancer cells, WHCO1 and WHCO6 using MTT cell viability assay 2. To investigate the effect of ?-Naphthoflavone on the expression of the CYP1 proteins (CYP1A1, CYP1A2 and CYP1B1) using Western blot analysis. 3. To determine whether the regulation of CYP1A1, CYP1A2 and CYP1B1 is associated with the AHR pathway by using Western blot analysis. 26 CHAPTER 2: The model 2.1. Introduction Cancer of the esophagus is the ninth most common cancer worldwide and recent evidence shows that its incidence is rising (Munoz, 1993; Parkin et al, 2001a; 2001b). Exposure to foreign chemi cal compounds is one of the main risk factors of this disease. Unfortunately, the hydrophobicity of these compounds is often an obstacle to their elimination. Hence, these compounds may accumulate to toxic levels, unless they are transformed to water soluble molecules that can be easily excreted. That is one of the reasons that Cytochrome P450 (CYP) enzymes were the chosen research topic since these enzymes play a major role in the oxidative metabolism of a wide range of toxic chemical compounds (Conney, 2003; Guengerich, 2004; Caro and Cederbaum, 2004; Coon, 2005; Ki m and Guengerich, 2005). A characteristic of CYP enzymes is their inducibility by chemical compounds thus allowing the cell to adapt to changes in its chemical environment. The aim of the current study is to determine the effect of ?-naphthoflavone (BNF), a synthetic derivative of a naturally occurring flavonoid, on the expression of CYP enzymes in moderately differentiated esophageal cancer cell lines, WHCO1 and WHCO6. First, an assessment of the BNF potency against the target cells was done in order to rule out the effect of toxicity. One of the obstacles in this assessment, however, was the poor solubility of BNF in the culture media; therefore, dimethyL sulfoxide (DMSO) was used to dissolve BNF. 27 DMSO was successfully used to increase the solubility of BNF, however, because DMSO has been reported to interact with cell membranes and affect metabolism in other cells (PenninCKx et al, 1983; Brayton, 1986), an experiment was done to determine the cytotoxic effects of DMSO alone on WHCO1 and WHCO6 cell lines. The 3-(4,5-Dimethylthiazol-2-yl)-2, 5- Diphenyltetrazolium bromide (MTT) assay was used to detect cytotoxicity or cell viability following exposure to DMSO according to the method of Mosmann et al, (1983). Briefly, the MTT assay measures the ability of viable cells to convert a soluble yellow tetrazolium salt (MTT) into insoluble purple formazan crystals by the mitochondrial dehydrogenase enzymes. The crystals are insoluble in aqueous solutions but can be dissolved in acidified isopropanol. The absorbance of the dissolved formazan solution is then quantified by measuring at a wavelength of between 500 and 600nm using a spectrophotometer. An increase in cell number leads to an increase in the amount of MTT formazan formed and an increase in absorbance (Mosmann et al, 1983). The aim was to determine the DMSO concentration with the least effect on the viability of WHCO1 and WHCO6 cells and subsequently dissolve BNF in that least toxic DMSO concentration. This study will also be of help in future to use these cell lines to study different types of proteins and their interaction in esophageal cancer. 28 2.2. Materials and Methods 2.2.1. Cell line and Cell Culture The human esophageal squamous carcinoma cell (HOSCC) lines WHCO1 and WHCO6 were derived from moderately differentiated squamous cell carcinoma tumors (Veale and Thornley, 1989). These cells were maintained in Dulbecco?s modified Eagle?s medium (DMEM) containing 4.5 g/L glucose, L- alanyl-glutamine, sodium bicarbonate and pyridoxine, HCl (Sigma), supplemented with 10% heat inactivated fetal bovine serum (FBS) (Sigma) at 37?C, 95% humidity and 5% CO 2. 2.2.2. Cell treatment WHCO1 and WHCO6 cells at 60-80% confluence were harvested and plated at a density of 5000 cells per well in 96-well flat-bottomed plates in a final volume of 90?l DMEM containing 10% FBS. The cells were left to grow at 37?C and 5% CO 2 atmosphere for 24 hours. The cells were then treated with DMSO at different concentrations (0%, 0.2%, 1%, 2.5%, and 5% v/v) in triplicate. To make a final volume of 100?l, DMEM (Sigma) containing 10% FBS was added. The cells were incubated at 37? C and a 5% CO2 atmosphere for 24 hours. 29 2.2.3. MTT Cell Viability Assay The MTT cell viability and proliferation assay was done by following the method previously used by Mosmann et al, (1983). To each well 10?l of MTT solution (5mg/ml) (Appendix 1.1) was a dded and the cells were incubated at 37?C for 4 hours. After 4 hours 100?l of the solubilisation reagent (10% SDS in 0.01M HCl) was added to each well and incubated at 37?C and a 5% CO 2 atmosphere for 16 hours. The absorbance was read with an iMarkTM microplate reader (Bio-Rad) at a wavelength of 590nm. The percentage of cell viability/DMSO cytotoxicity was calculated relative to control cells designated as 100% viable cells. This experiment was repeated 3 times. 2.2.4. Statistical Analysis The results were expressed as means ? standard deviations (SD) for at least three independent experiments. Significant differences were determined using one way analysis of variance (ANOVA) followed by Tukey?s post comparison test (Graphpad prism 5) (Appendix 2-3). A probability level of P<0.05 was considered significant. 30 2.3. Results MTT cell viability test was used to determine the effect of DMSO on the viability of two esophageal cancer cell lines WHCO1 and WHCO6 after 24- hour incubation. Figure 2.1 shows the percentage cell viability in relation to DMSO concentration after 24-hour incubation. The results showed a gradual decrease of cell number with an increase in DMSO concentration. It was also found that there was a significant difference (P<0.05) in the toxic effect of DMSO at concentrations from 0-5% (v/v). At higher DMSO concentrations, the number of cells decreased drastically when compared to the lower DMSO concentrations. Figure 2.1 below shows the effects of varying concentrations of DMSO on the two cell lines, WHCO1 and WHCO6. 31 A B Figure 2.1 : The effect of DMSO on cell viability. WHCO1 (A) and WHCO6 (B) cells were incubated with increasing concentrations of DMSO for 24 hours. Cell viability was measured by the MTT assay and the percentage of viable cells relative to the control was calculated for each concentration. Error bars represent the mean ? S.D. from three i ndependent experiments. Statistical differences were determined with one-way analysis of variance (ANOVA) followed by Tukey?s multiple comparison test (Graph pad prism 5). 32 2.4. Discussion and Conclusion Compounds that are poorly soluble in water or culture media are often encountered during the process of drug discovery. Solvents are thus used to increase the solubility of such compounds. DMSO is an amphipathic molecule consisting of a polar domain and two non-polar methyl groups thus making it to be easily soluble in both aqueous and organic media. DMSO is one of the most common solvents that have been used to dissolve hydrophobic compounds. However previous studies have shown DMSO to penetrate through cells, thus influencing the structure and conformation of proteins and cell membrane, thus affecting cell viability (PenninCKx et al, 1983; Brayton, 1986). Therefore, in experiments whereby DMSO is to be used as a solvent, it is always important to determine the effect of only the solvent and use a correction factor when the toxicity of another compound is calculated. DMSO was used as a solvent to dissolve the compound of interest BNF. In order to determine what concentration of DMSO was suitable to dissolve BNF and had least effect on the viability of WHCO1 and WHCO6 cells, different concentrations of DMSO were added to the two cell lines and the effect was examined using the MTT cell viability test. The effects of DMSO on cell viability have been studied in a large number of cell types, but variable results have been reported in these studies. For example, most studies have shown insignificantly lower or no effect on cell 33 viability after 24- hour treatment with 0.1% DMSO (Skupinska et al, 2007; Maruyama et al, 2007; Tampio et al, 2008). Studies with lymphoma cells have shown DMSO concentrations of 1-2% to prevent apoptosis (Lin et al, 1995). Other cell types appear to be more resistant to the cytotoxic effects of DMSO, for example, DMSO concentration of 10% had no cytotoxic effects on Caco2/Tc7 cells (Da Violante et al, 2002). In this study, 5% (v/v) DMSO which is the highest DMSO concentration which was used was found to have high cytotoxic effects when compared to the other concentrations, causing a reduction of cell viability to 12% in WHCO1 and 40% in WHCO6 cells (Figure 2.1). Only treatment with 0.2% v/v DMSO gave similar percentage viability to that of the control cells in both cell lines. However, DMSO was found to be more toxic in WHCO1 cells than in WHCO6 cells in all the different concentrations , this can be seen on figure 2.1 where the less toxic DMSO concentration, 0.2% (v/v), redu ced cell viability to 90% in WHCO1 cells (Figure 2.1A) and 98% in WHCO6 cells (Figure 2.1B). These results are in agreement with previous studies which also showed 0.2% (v/v) DMSO to be a safe concentration for use in tissue culture since DMSO at this concentration seems to cause less or no effect in cell viability (Hukkanen et al, 1999; Gelardi et al, 2001; Nishi et al, 2002). In summary, the results observed here indicate that 24 hour treatment with DMSO concentrations of 5% (v/v) have higher cytotoxic effects on esophageal cancer cell lines WHCO1 and WHCO6 , with WHCO1 cells being more 34 sensitive to the cytotoxic effects of DMSO at both higher and lower DMSO concentrations when compared to the WHCO6 cells. The results show that at concentrations up to 0.2% (v/v), DMSO had no cytotoxic effects on WHCO1 and WHCO6 cells; hence, BNF was di ssolved in 0.2% DMSO for use in subsequent experiments. Although concentrations lower than 0.2% were shown to be associated with much reduced toxicity, they were not good in dissolving the required drug. 35 CHAPTER 3: The value of BNF as an antiproliferative agent for esophageal cancer cells 3.1. Introduction Prognosis of esophageal cancer is poor with an overall 5-year survival rate of less than 10%. There are many factors that have been associated with increased risk of developing esophageal cancer and these include, gender, ethnicity, environmental factors as well as genetic factors. Therefore, early detection and search for potential anticancer compounds is important in the control of this carcinoma. Studies have revealed a large variety of phyto-chemicals that have proven successful against a wide range of cancers. In particular, flavonoids have been shown to influence a variety of biological functions including inhibition of cancer cell growth (Marchand et al, 2000). The chemical structure of flavonoids is derived from a heterocyclic hydrocarbon, chromane, by substituting its ring C with phenyl group (ring B) in either position 2 or 3 (Gary, 2003) (Figure 3.1). Flavonoids are classified as flavonols, flavones, flavanols, flavanones, anthocyanidins and isoflavonoids based on the differences in the chemical structure of the heterocyclic C ring, the number and position of the double bonds and hydroxyl(OH) groups (Kozikowski et al, 2003). It has been reported that flavonoids with 4-6 OH groups often act as strong antioxidants, while those with more or fewer OH 36 groups show low or no antioxidant activities (Rice-Evans et al, 1995; Rice- Evans et al, 1996). Figure 3.1 : Different types of flavonoid structures (adapted from Hodek et al, 2002). Compounds of flavonoid structure have shown their antiproliferative effects by delaying or reversing the process of carcinogenesis. They achieve this by protecting membranes, proteins and DNA from oxidative damage, by 37 scavenging hydroxyl radicals, superoxide anion radicals and lipid peroxyradicals (Chan et al, 1998, Doostdar et al, 2000, Henderson et al, 2000). They also inhibit enzymes implicated in procarcinogenesis and detoxification of xenobiotics (Bravo, 1998). Considering the antiproliferative activity of some flavonoids on certain tumor cells and due to lack of such knowledge on esophageal cancer cells, in the current study the antiproliferative activity of a synthetic derivative of a naturally occurring flavonoid, ?-Naphthoflavone (BNF) on WHCO1 and WHCO6 cells was evaluated. This study may provide some new knowledge about esophageal cancer therapy. 38 3.2. Materials and Methods 3.2.1. Cell line and Cell Culture WHCO1 and WHCO6 cells were maintained by following the protocol described in chapter 2 (Section 2.2.1) 3.2.2. Cell treatment At least three independent studies were conducted in triplicates each time. WHCO1 and WHCO6 cells at 60-80% confluence were harvested and plated at a density of 5000 cells per well in 96-well flat-bottomed plates. ?- Naphthoflavone (ANF) and Benzo pyrene (BP) were used as negative controls and 3-Methylcholanthrene(3MC) was used as a positive control in order to assess the validity of the MTT cytotoxicity assay. Stock solutions of BNF, ANF, BP and 3MC (50 Mm and 10 Mm) were dissolved in DMSO and added to the media to give final concentrations of 5, 10, 25, 50 and 100 ?M BNF (in each case the final concentration of DMSO in media was 0.2%) in a final volume of 100 ?l. Cells treated with the solvent alone (0.2% DMSO) also acted as the negative control.. The cells were treated with BNF, ANF, BP and 3MC at these concentrations for 24, 48 and 72 hours and incubated at 37?C, 95% humidity and 5%CO2 39 3.2.3. MTT Cell Viability Assay After the incubation period, 10?l of the MTT (final concentration of 0.5 mg/ml) (Appendix 1.1) was added to each well and incubated for 4 hours at 37?C, 95% humidity and 5% CO 2. 100?l of the solubilization solution (10% SDS in 0.01M HCL) was added to each well. The plate was incubated overnight at 37?C, 95 % humidity and 5% CO2. The absorbance of the formazan produced by the viable cells was measured for each well at a wavelength of 590nm using an iMARKTM microplate reader (Bio-Rad). The percentage of cell viability was calculated relative to control cells (0.2% DMSO treated cells) designated as 100% viable cells. 3.2.4. Determining the median in hibition concentration (IC ) 50 To assess the effectivity of a compound in inhibiting cell proliferation, an IC50, which is a concentration at which 50% of cell growth is inhibited, is usually calculated. The IC50 for BNF was calculated after treatment in WHCO1 and WHCO6 cells (Appendix 6). 3.2.5. Statistical Analysis The results were expressed as means ?standard deviations (S.D) for at least three independent experiments. Significant differences were determined using one-way analysis of variance (ANOVA) followed by Tukey?s multiple 40 comparison test (Graphpad prism 5) (Appendix 4-5) A probability level of p<0.05 was considered significant. 41 3.3. Results 3.3.1. The antiproliferative activity of BNF against WHCO1 and WHCO6 esophageal cancer cells To evaluate the effect of BNF, WHCO1 and WHCO6 cells were treated with increasing concentrations of BNF for 24, 48 and 72 hours. Cell viability was measured using the MTT assay. To assess the validity of the MTT cell viability assay BP and ANF were used as negative controls and 3MC was used as a positive control. The assay lasted for 72 hours because other situations may arise at longer incubation periods, such as quiescence, metabolic stopping or induction of apoptosis. BNF showed the biggest antiproliferative effects against WHCO1 and WHCO6 esophageal cancer cells which was dose and time dependent (see Figure 3.2 and 3.3). However, the effects of BNF seemed to diminish overtime in the WHCO1 cell while in the WHCO6 cell line the effects increased with time. The 3MC, used as a positive control in this study was associated with a significant (P< 0.05) reduction in pro liferation of both WHCO1 and WHCO6 and its effects were also time and dose dependent. However, compared to the effect of BNF, 3MC was less potent against the proliferation of both WHCO1 and WHCO6 cells. 42 BP and ANF were used as negative controls. These two compounds seemed not to have any activity against the proliferation of both WHCO1 and WHCO6 cells. The results demonstrate that with increasing concentrations of BP and ANF from 5?M to 25?M the percentage of cell proliferation increased progressively after 24-48 hour of exposure. It was only after 72h of 100?M BP and ANF exposure that cell proliferation was inhibited to below 80% (Figure 3.3). 24H Treatment WHCO1 0 20 40 60 80 100 120 140 0 5 10 25 50 100 Concentration(uM) C el l pr ol ife re tio n( % ) BP BNF 3MC ANF 24H Treatment WHCO6 0 20 40 60 80 100 120 140 0 5 10 25 50 100 Concentration(uM) C el l p ro lif er at io n( % ) BP BNF 3MC ANF Figure 3.2: The effect of BNF on the proliferation of human esophageal carcinoma cells (WHCO1 and WHCO6) after 24 hours. At least three independent studies were conducted in triplicates each time. Cells were plated at a density of 5000 cells/well in a 96 well plate and incubated with increasing concentrations of BNF for 24 hours. BP and ANF were used as negative controls and 3MC as a positive control. Cell proliferation was measured by the MTT assay and the percentage of viable cells relative to the control (cells treated with DMSO only) was calculated for each concentration. Results are presented as the means ? SD from three independent experiments. Statistical differences were determined with the one-way ANOVA followed by Tukey?s post comparison test. P<0.05 was considered significant. 43 48H Treatment WHCO1 0 20 40 60 80 100 120 0 5 10 25 50 100 Concentration(uM) C el l pr ol ife ra tio n( % ) BP BNF 3MC ANF 48H Treatment WHCO6 0 20 40 60 80 100 120 140 0 5 10 25 50 100 Concentration(uM) C el l p ro lif er at io n( % ) BP BNF 3MC ANF 72H Treatment WHCO1 0 20 40 60 80 100 120 0 5 10 25 50 100 Concentration(uM) C el l pr ol ife ra tio n( % ) BP BNF 3MC ANF 72H Treatment WHCO6 0 20 40 60 80 100 120 140 0 5 10 25 50 100 Concentration(uM) C el l p ro lif er at io n( % ) BP BNF 3MC ANF Figure 3.3: The effect of BNF on the proliferation of human esophageal carcinoma cells (WHCO1 and WHCO6) after 48 and 72 hours. At least three independent studies were conducted in triplicates each time. Cells were plated at a density of 5000 cells/well in a 96 well plate and incubated with increasing concentrations of BNF for 48 and 72 hours. BP and ANF were used as negative controls and 3MC as a positive control. Cell proliferation was measured by the MTT assay and the percentage of viable cells relative to the control (cells treated with DMSO only) was calculated for each concentration. Results are presented as the means ? SD from three independent experiments. Statistical differences were determined with the one-way ANOVA followed by Tukey?s post comparison test. P<0.05 was considered significant. 44 3.3.2. The median inhibition concentration (IC 50) values of BNF in WHCO1 and WHCO6 cells. For a compound to be considered as anticancerous it must display the desired level of inhibitory activity against the target cell type. Activity is expressed as the median inhibition concentration (IC50) which is the concentration at which a given test compound inhibits cell proliferation by 50%. The 50% proliferation was calculated relative to control cells (0.2% DMSO treated cells) designated as 100% viable cells. Figure 3.4 shows the effects of varying BNF concentrations on the two cell lines. For the WHCO1, to reach 50% toxicity, a concentration of 25 uM was required while for the WHCO6, 50% toxicity was reached with the use of 10 uM BNF. BNF resulted in IC50 after treating WHCO1 cells with 25?M BNF and incubating them for 24 hours while 10?M BNF resulted in IC50 in the WHCO6 after incubating them for 48 hours period. 45 Figure 3.4: The median inhibition concentration (IC50) of BNF in WHCO1 and WHCO6 cells. IC50 is a concentration at which 50% of cell growth is inhibited. The red dotted line shows 50% cell growth inhibition. Results represent means ? SD of at least 3 different experiments conducted in triplicates each time. P<0.05 is considered significant 46 3.4. Discussion and Conclusion Flavonoids are polyphenolic compounds occurring in plants. They have been found in dietary components such as fruits, vegetables, and tea (Barnes et al, 2001; Liu, 2003). Besides their role in plants, these compounds have shown to reduce the risk of cancer development and other major chronic diseases (Marchand et al, 2000). ?-Naphthoflavone (BNF) is a synthetic derivative of a naturally occurring flavonoid. The antitumor effects of BNF have been investigated only recently. This flavonoid has been shown to possess antiproliferative effects in breast cancer cells (MCF7) (C?rdenas et al, 2006). However, there are few reports documenting the anticancerous activities of BNF in other cancer cell types. Therefore, in the current study the antitumor activities of BNF in human esophageal cancer cells, WHCO1 and WHCO6 cells were further explored. BNF exerted significant antiproliferative effects against both cancer cell lines. The antiproliferative effects were expressed as the median inhibition concentration (IC ) values. Based on the IC50 50 values, compounds are usually classified as either highly active (IC < 10?M) or moderately active (IC50 50~10- 20?M) (C?rdenas et al, 2006). The magnitude of the antiproliferative effects of BNF were different in the two cell lines, WHCO6 responded very well to low BNF concentrations while WHCO1 cells needed a higher concentration of BNF. Interestingly, BNF effect diminished over time in WHCO1 cells while in 47 WHCO6 cells the effect increased. BNF caused moderate antiproliferation in WHCO6 (IC =10?M) but weak effect in WHCO1 cells (IC50 50 =25?M).The reason for the differences in response to BNF in both WHCO1 and WHCO6 could be due to the contribution of metabolizing gene polymorphisms in the two cell lines. These results therefore support the observation that cancerous tumors usually contain cell subpopulations with different biological properties and suggest a differential selective action in different cell types. The effect of BNF on proliferation was compared to some of the well studied compounds, BP, 3MC and ANF. BP, a polyaromatic hydrocarbon (PAH) which is commonly present in tobacco smoke has been implicated in the induction of cell proliferation (Jeffy et al, 1999, Culp et al, 2000; Jeffy et al, 2002). To exert its adverse health effects, BP requires metabolic activation to a biologically reactive intermediate. The cell proliferation effect of BP has been studied in several cell lines, thus this hydrocarbon together with ANF, one of the most common CYP1 inhibitors (Gasiewicz and Rucci, 1991; Gasiewicz et al, 1996) were used as negative controls in this study. The data clearly showed that both BP and ANF did not have detectable antiproliferative effects in both WHCO1 and WHCO6 but instead, treatment of cell lines with these compounds resulted in significantly increasing cell proliferation even at low concentrations. These results are in agreement with previous findings that have shown BP to enhance proliferation of human breast cancer cells and lung cancer cells (Tsai et al, 2004; Kometani et al, 2009). 48 3MC which is one of the well known CYP1 classical inducers was shown to have antiproliferative effects in both WHCO1 and WHCO6 cell lines. This type of behavior was also reported in previous studies whereby 3MC displayed antiproliferative effects in human keratinocyte cell line NCTC 2544 (Gelardi et al, 2001), rat calvarial osteoblast-like cells and mouse calvarial clonal preosteoblastic cells (MC3T3-E1) (Naruse et al, 2002). The results also showed that BNF was associated with significant antiproliferative effects on esophageal cancer cells and thus it would be suggested that esophageal cancer cells may be responsive to the treatment with BNF. However, further investigations in this direction are required. 49 CHAPTER 4: The effects of BNF on the expression of CYP1A1, CYP1A2, CYP1B1 and AHR in the two esophageal cancer cell lines, WHCO1 and WHCO6 4.1. Introduction The human cytochrome P450 (CYP) 1 gene family is one of the main CYP families involved in the metabolism of a wide range of xenobiotics (Gonzalez and Gelboin, 1994; Shimada et al , 1996; 1997; Crespi et al, 1997) and consists of three known enzymes, CYP1A1, CYP1A2 and CYP1B1. The inducible expression of the CYP1 family is regulated via the aryl hydrocarbon receptor (AHR) complex (Hankinson, 1995; Schm idt and Bradfield, 1996). In its inactive form, the AHR is located in the cytosol where it is bound to a dimmer of heat shock proteins (HSP90), one molecule of p23 and one molecule of the AHR interacting protein (AIP) (Hollingshead et al, 2004; Bock and Kohle, 2006). Upon ligand binding the AHR dissociates from the proteins and forms a ligand-AHR complex. This complex is translocated to the nucleus and forms a heterodimer with the aryl nuclear translocator (ARNT) (Fujii-Kuriyama and Mimura, 2005). This heterodimer binds to the xenobiotic response element and activates the transcription of numerous genes, including CYP1A1, CYP1A2, and CYP1B1 (Hankinson, 1995; Mimura and Fujii-Kuriyama, 2003). 50 This chapter describes the experiments that were carried out using Western blot analysis to investigate the effect of BNF on the expression of individual members of the CYP1 gene family in WHCO1 and WHCO6 esophageal cancer derived cell lines. The expression of these forms of CYPs was compared to that of the AHR. 51 4.2. Materials and Methods 4.2.1. Cell Culture and Treatment WHCO1 and WHCO6 cells were maintained by following the protocol described in chapter 2 (Section 2.2.1). At 60-80% confluence the cells were rinsed with 1X phosphate buffer saline (PBS), (136.8 mM NaCl, 2.68 mM KCl, 10.1 mM Na2HPO4?12H 2O, 1.76 mM KH2PO4 pH 7.2), trypsinized with trypsin/ EDTA (0.01% trypsin, 0.004% EDTA) and seeded into10 cm tissue culture dishes (Falcon). After 24 hours the cells were rinsed with 1X PBS and treated with BNF(WHCO1 cells treated with 25?M BNF for 24 hours and WHCO6 cells treated with 10?M BNF for 48 hours) and incubated at 37?C, 95% humidity and 5% CO2. Control cells were treated with 0.2% DMSO. Whole cell extractions of the treated and control cells were performed after the incubation period of each cell line. 4.2.2. Whole Cell protein Extraction Cells were rinsed three times with ice-cold PBS and scraped off the dish in a final volume of 1ml PBS and transferred into a sterile eppendorf tube. The cells were centrifuged at 1145 x g for 2 minutes using the bench top mini centrifuge. The supernatant was discarded and the pellet resuspended in 100?l Laemmli sample buffer (Appendix 7.1). The samples were boiled for 5 minutes followed by centrifugation at 10 000g for 10 minutes at 4?C. The samples were then stored at -70?C. 52 4.2.3. Protein Estimation The protein concentrations of the cell extracts were determined using the method proposed by Bramhall et al, 1969. This method is described below. A 15cm filter paper (Whatman) was rinsed in distilled water for 20 minutes. This was followed by rinsing in 95% ethanol for 5 minutes, 99% ethanol for 5 minutes, acetone for 5 minutes, consecutively, in order to dehydrate the filter paper. The filter paper was then left under a fume hood to dry. Bovine serum albumin (BSA) was dissolved in Laemmli sample buffer (Appendix 7.1) to make a final concentration of 2?g/?l. A range of protein standards with a final concentration of between 1 and 20?g BSA were prepared and loaded onto the dry filter paper. Five (5) ?l of each protein sample was also loaded onto the filter paper. The filter paper was left under the fume hood to dry. The BSA standards and the protein samples were fixed within the filter paper by rinsing the filter paper with 7.5% tichloro-acetic acid (TCA) (Appendix 7.2) for 40 minutes and stained for 45 minutes with 0.25% coomassie blue stain solution (Appendix 7.2). The filter paper was destained in destaining solution (Appendix 7.2) until only the stained protein spots and BSA standards sports remained. The filter paper was left to dry and the stained spots were individually cut and placed in 5ml elution solution (Appendix 7.2) overnight in the dark. The absorbance of each sample was measured at 596nm. The BSA standards absorbance values were used to construct a standard curve. The concentration of each protein sample was calculated from the standard curve. 53 4.2.4. SDS-PAGE The SDS-PAGE method used was adapted from Laemmli, 1970. A 10% separating gel (Appendix 7.3) and 5% stacking gel (Appendix 7.3) were prepared. Each sample was loaded to the gel at the required volume to obtain 35?g of protein for the detection of CYP1A1, 25?g for the detection CYP1A2, 15?g for the detection of CYP1B1 and 10?g for the detection of AHR. Protein molecular weight marker (Fermentas) was also loaded. The gel was electrophoresed at a constant current of 25 milli-Amps (mA) in running buffer (Appendix 7.3). 4.2.5. Western Blotting For the detection of 56kDa CYP1A1, CYP1A2, and CYP1B1 as well as 122kDa AHR proteins, the proteins were electrically transferred from the gel onto the nitrocellulose membranes, in transfer buffer (Appendix 7.4) for 1 hour at a constant current of 400mA using the Biorad mini trans-blot system. The membranes were rinsed 3 times with PBS. To block non-specific proteins, the membranes were incubated for 1 hour in 5% non-fat milk powder in Tris buffered saline (TBS) or 5% BSA in Tris buffered saline containing 0.1% Tween-20(TBST). The membranes were rinsed 6 times with PBS or TBST and incubated overnight at 4?C with rabbit anti-CYP1A1 antibody (1:1000, SANTA CRUZ), rabbit anti-CYP1A2 (1 :1500, SANTA CRUZ), rabbit anti- CYP1B1 (1:1000, SANTA CRUZ) or rabbit anti AHR antibody (1:800, 54 SANTA CRUZ) in 1% BSA. After 6 X 5 minutes rinsing in PBS or TBST the membranes were incubated with 1:5000 horseradish peroxidase conjugate (HRP) goat anti-rabbit antibody (Sigma) for an hour in 1% BSA or 5% non-fat milk powder at room temperature in the dark. The membranes were then rinsed for a further 30 minutes in PBS or TBST at 5-minute intervals. The super signal west pico chemiluminescent substrate kit (Pierce) (Appendix 7.4) was used for the detection of of the proteins. The membranes were then exposed to the hyperfilm for 10 minutes. The film was developed for 5 minutes in the developer (Appendix 7.4), rinsed with water and placed in fixer (Appendix 7.4) for 5 minutes and finally rinsed with water. 4.2.6. Densitometry Densitometric methods are used to measure the relative quantities of proteins on the blot compared to the control proteins. LabWorksTM Image Acquisition and analysis Software (LabWorks version 4.5) was used to quantitatively determine the concentration level of CYP1A1, CYP1A2, CYP1B1 and AHR. 4.2.7. Statistical Analysis The results were expressed as means ?standard deviations (S.D) for at least three independent experiments. Significant differences between the control cells and BNF treated cells were determined using the T-test (Excel 2003) (Appendix 8). A probability level of p<0.05 was considered significant. 55 4.3. Results 4.3.1. SDS-PAGE for protein estimation To compare a change in expression between treated and non- treated samples using Western blotting the same concentration of both samples was used. Each sample was loaded to the gel at the required volume to obtain 15?g of protein. Figure 4.1 shows SDS-PAGE of samples extracted from WHCO1 and WHCO6 cell lines. The uniform band intensities obtained for background protein for all samples analyzed by SDS-PAGE (Figure 4.1), is confirmation that similar concentrations of cell lysate were loaded in each well. 56 M 1 2 3 4 170 kDa Figure 4.1 SDS-PAGE of samples extracted from WHCO1 and WHCO6 cell lines. Whole cell protein extraction was used and the total protein was estimated as explained in materials and methods. 15?g of each sample was loaded in each lane. Lanes- M: Marker, Lane 1: WHCO1 control, Lane 2: WHCO1 BNF treated, Lane 3: WHCO6 control, Lane 4: WHCO6 BNF treated. 4.3.2. The effect of BNF on CYP1A1, CYP1A2, CYP1B1 and AHR protein expression To examine the effect of BNF on the expression of CYP1A1, CYP1A2, CYP1B1 and AHR, both WHCO1 and WHCO6 cells were treated with their respective BNF IC50 concentrations whereby the WHCO1 cells were treated with 25?M BNF for 24 hours and WHCO6 cells were treated with 10?M BNF for 48hours. The expression of these proteins was measured using Western blot analysis. Fold increases or decreases in protein expression was determined by 130 kDa 35 kDa 25 kDa 100 kDa 70 kDa 55 kDa 40 kDa 57 comparing protein levels of CYP1A1, CYP1A2, CYP1B1 and AHR in BNF treated cells to protein levels of each respective gene in control cells by using the densitometric band intensities of Western blots. 4.3.2.1. The effect of BNF on CYP1A1 protein expression The CYP1A1 protein was constitutively expressed in both WHCO1 and WHCO6 cell lines. Treatment of these cells with BNF resulted in an increase in CYP1A1 protein expression that was significantly different (Appendix 8) from the controls. WHCO1 cells treated with BNF displayed a 1.3 fold increase in CYP1A1 protein expression compared to control cells (P=0.02) (Figure 4.2) while WHCO6 cells treated with BNF displayed a 1.1 fold increase in CYP1A1 protein expression compared to control cells (P=0.01). 58 WHCO6 WHCO1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Control BNF C Y P 1A 1 p ro te in E xp re ss io n (F o ld in d u ct io n ) 56kDa 56kDa 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Control BNF C Y P 1A 1 P ro te in E xp re ss io n ( F o ld in d u ct io n ) Figure 4.2 The effect of BNF on the expression of CYP1A1 protein in WHCO1 and WHCO6 cells. Representative Western blots are shown in this figure. WHCO1 and WHCO6 cells were exposed to 25?M BNF for 24 hours and 10?M BNF for 48 hours, respectively, while control cells were treated with 0.2% DMSO. 35?g of the whole cell protein extract of each sample was loaded in each lane. The transferred proteins were probed with rabbit anti-CYP1A1 antibody followed by (HRP) goat anti-rabbit antibody. The densitometric band intensities of Western blots are expressed as fold change relative to the control. Statistical differences were determined by using the T-test whereby P< 0.05 was considered significant. 4.3.2.2. The effect of BNF on CYP1A2 protein expression Similar to the CYP1A1, CYP1A2 protein was also discovered to be constitutively expressed in WHCO1 and WHCO6 cells. The treatment of these cells with BNF displayed an increase in CYP1A2 protein expression as shown on figure 4.3. The change in CYP1A2 protein expression was 59 significantly increased to approximately 1.4 fold over control cells in both cell lines (P=0.01 in WHCO 1, P=0.05 in WHCO6). WHCO6 WHCO1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Control BNF C Y P 1A 2 P ro te in E xp re ss io n (F o ld in d u ct io n ) 56kDa 56kDa 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Control BNF C Y P 1A 2 P ro te in e xp re ss io n (F o ld in d u ct io n ) Figure 4.3 The effect of BNF on the expression of CYP1A2 protein in WHCO1 and WHCO6 cells. Representative Western blots are shown in this figure. WHCO1 and WHCO6 cells were exposed to 25?M BNF for 24 hours and 10?M BNF for 48 hours, respectively, while control cells were treated with 0.2% DMSO. 25?g of the whole cell protein extract of each sample was loaded in each lane. The transferred proteins were probed with rabbit anti-CYP1A2 antibody followed by (HRP) goat anti-rabbit antibody. The densitometric band intensities of Western blots are expressed as fold change relative to the control. Statistical differences were determined by using the T-test whereby P< 0.05 was considered significant. 4.3.2.3. The effect of BNF on CYP1B1 protein expression Figure 4.4 shows the effect of BNF on the expression of CYP1B1 protein. The CYP1B1 constitutive expression was very low in both cell lines, however, significant inductions in CYP1B1 protein expression were observed after BNF 60 treatment. WHCO1 cells treated with BNF showed a 2.4 fold increase in CYP1B1 protein expression compared to control cells (P=0.003) (Figure 4.4) while WHCO6 cells treated with BNF displayed a 1.7 fold increase in CYP1B1 protein expression compared to control cells (P=0.05) (Figure 4.4). WHCO6 WHCO1 0 0.5 1 1.5 2 2.5 3 Control BNF C Y P 1B 1 P ro te in e xp re ss io n (F o ld in d u ct io n ) 0 0.5 1 1.5 2 2.5 3 Control BNF C Y P 1B 1 P ro te in E xp re ss io n (F o ld in d u ct io n ) 56kDa 56kDa Figure 4.4 The effect of BNF on the expression of CYP1B1 protein in WHCO1 and WHCO6 cells. Representative Western blots are shown in this figure. WHCO1 and WHCO6 cells were exposed to 25?M BNF for 24 hours and 10?M BNF for 48 hours, respectively, while control cells were treated with 0.2% DMSO. 15?g of the whole cell protein extract of each sample was loaded in each lane. The transferred proteins were probed with rabbit anti-CYP1B1 antibody followed by (HRP) goat anti-rabbit antibody. The densitometric band intensities of Western blots are expressed as fold change relative to the control. Statistical differences were determined by using the T-test whereby P< 0.05 was considered significant. 61 4.3.2.4. The effect of BNF on AHR protein expression Treatment of WHCO1 and WHCO6 cells with BNF caused a down regulation in AHR protein expression that was significantly different (Appendix 8) from the controls. WHCO1 cells treated with BNF caused a 1.3 fold decrease in AHR protein expression compared to the control cells (P=0.003) (Figure 4.5). With regard to the WHCO6 cells, BNF caused a 4.5 fold decrease in AHR protein expression compared to the control cells (P=0.004) (Figure 4.5). WHCO1 WHCO6 0 0.2 0.4 0.6 0.8 1 1.2 Control BNF A H R P ro te in e xp re ss io n (F o ld in d u ct io n ) 122kDa 122kDa 0 0.2 0.4 0.6 0.8 1 1.2 Control BNF A H R P ro te in E xp re ss io n (F o ld in d u ct io n ) Figure 4.5: The effect of BNF on the expression of AHR protein in WHCO1 and WHCO6 cells. Representative Western blots are shown in this figure. WHCO1 and WHCO6 cells were exposed to 25?M BNF for 24 hours and 10?M BNF for 48 hours, respectively, while control cells were treated with 0.2% DMSO. 10?g of the whole cell protein extract of each sample was loaded in each lane. The transferred proteins were probed with rabbit anti-AHR antibody followed by (HRP) goat anti-rabbit antibody. The densitometric band intensities of Western blots are expressed as fold change relative to the control. Statistical differences were determined by using the T-test whereby P< 0.05 was considered significant. 62 4.3.3. BNF induces the expression of CYP1B1 protein to significantly higher levels than CYP1A1 and CYP1A2 proteins in WHCO1 and WHCO6 cells To compare the relationship amongst CYP1A1, CYP1A2 and CYP1B1 protein expression in response to BNF treatment in WHCO1 and WHCO6 cells, the ratios of CYP1B1 to CYP1A1 fold changes (CYP1B1 fold change/CYP1A1 fold change), CYP1B1 to CYP1A2 fold changes (CYP1B1 fold change/CYP1A2 fold change), and CYP1A2 to CYP1A1 fold changes (CYP1A2 fold change/CYP1A1 fold cha nge) were calculated for each cell line (Table 4.1). Ratios that are greater than one, for example in terms of the CYP1B1 to CYP1A1 fold change, would mean that the expression of CYP1B1 was induced to a higher level than the expression of CYP1A1 protein (Table 4.1). Similar conclusions were made for the other protein ratio changes. Table 4.1: Comparison of CYP1 A1, CYP1A2 and CYP1B1 expression in BNF treated cells. Dose (?M)) Time (h) Cell line CYP1B1/1A1 ratio CYP1B1/1A2 ratio CYP1A2/1A1 ratio WHCO1 25 24 2.2 1.7 1.1 WHCO6 10 48 1.6 1.2 1.3 Ratios that are greater than one (1) show a higher level of induction 63 4.4. Discussion 4.4.1. BNF induces the exp ression of CYP1 enzymes Flavonoids are a large class of phenolic compounds present in fruits, vegetables and other plant foods and have been shown to reduce the risk of cancer and other major chronic diseases. They show their effects by either delaying or reversing the process of carcinogenesis. At the first entry site, procarcinogens are metabolized in phase I reactions catalyzed by CYP enzymes (Conney, 2003; Guengerich, 2004; Ca ro and Cederbaum, 2004; Coon, 2005; Kim and Guengerich, 2005) and are then conjugated with water soluble endogenous metabolites by phase II enzymes, thus producing hydrophilic products that can be easily excreted (Burchell, 2003; Kuuranne et al, 2003). However, phase I products are not always conjugated by phase II enzymes since the phase I products are often highly reactive metabolites capable of causing toxicity (Guengerich, 2000; Schwarz et al, 2001). Flavonoids have been shown to interact with CYP enzymes in at least three ways: they induce the biosynthesis of several CYPs, they modulate (inhibit or stimulate) the activities of CYP enzymes and flavonoids are also metabolized by several CYPs (Hodek et al, 2002). Some of the beneficial properties of flavonoids include inhibition of CYPs involved in carcinogen activation and scavenging of reactive radicals formed from carcinogens by CYP-mediated reactions (Chan et al, 1998; Zhai et al, 1998; Doostdar et al , 2000; Henderson 64 et al, 2000). Flavonoids can also prevent the process of carcinogenesis by induction of phase II enzymes as well. In the current study, the inducible expression of the enzymes (CYP1A1, CYP1A2 and CYP1B1) in response to treatment of esophageal cancer cells, WHCO1 and WHCO6 by the synthetic flavonoid BNF was investigated. An induction of CYP1A1, CYP1A2 and CYP1B1 by BNF is reported in this study. This flavonoid was shown to induce CYP1A1 by 1, 3 and 1, 1 fold in WHCO1 and WHCO6 cells, respectively. It was also shown to induce CYP1A2 by 1, 4 fold in both cell lines. In addition, this flavonoid also induced CYP1B1 by 2.4 and 1, 7-fold induction in WHCO1 and WHCO6 cells, respectively. Although CYP enzymes generally convert xenobiotics to less toxic compounds, the reactions frequently involve the formation of reactive intermediates capable of causing toxicity. Elevation of activities of CYP1 family of enzymes (CYP1A1, CYP1A2 and CYP1B1) is highly unwanted since these enzymes are responsible for activation of carcinogens such as BP, 7, 12-dimethylbenz (a) anthracene (DMBA), Aflatoxin B1 and meat derived heterocyclic aromatic amines (Omiecinski et al, 1999). CYP1 induction can also cause an imbalance between phase I and phase II reactions, for example when CYP1 enzymes are more active than phase II enzymes, this can cause an accumulation of phase I products capable of causing toxicity (Guengerich, 2000; Schwarz et al, 2001). CYP1 enzymes have broad substrate specificities 65 therefore induction of these enzymes may result in alteration of pharmaceutical drugs thus causing an overdose or loss of their therapeutic effect (Tang and Stearns, 2001; Rochat et al, 2001; Mcfadyen et al, 2001; Bournique and Lemarie, 2002). The induction of CYP1 enzymes is recognized as a factor in determining the risk of the development of cancer. Increased expression of CYP1A1 in the lungs increases the risk of lung cancer (McLemore et al, 1990; Guengerich, 1988) as well as colorectal cancer (Sivaraman et al, 1994). CYP1A2 also has a role in the development of cancers associated with tobacco smoking. Differential expression of the CYP1 enzymes was observed in WHCO1 and WHCO6 cells, with CYP1B1 induction being the highest compared to the other enzymes after BNF exposure. CYP1A1 was the second highly induced enzyme while CYP1A2 was induced the least after BNF exposure. These results are in agreement with previous results that showed CYP1B1 enzyme to be more active than CYP1A enzymes in metabolizing several compounds to reactive intermediates (Shimada et al, 1996; Li et al, 1998; Yengi et al, 2003; Wen and Walle, 2005; Walle et al, 2006). CYP1B1 induction can thus be used as a target in cancer therapy and prevention, especially since this enzyme has been shown to be up-regulated in a wide variety of cancers (Murray et al, 1997; Ko et al, 2001; Murray et al , 2001; McFadyen et al, 2001; Tanaka et al, 2002; Chang et al, 2003; Carnell et al, 2004; Wen and Wa lle, 2005; Walle et al, 2006). 66 When comparing the ratio of protein level in both WHCO1 and WHCO6 cells, the induction of the CYP1 enzymes was higher in WHCO1 than in WHCO6 cells. These results therefore suggest that the ability of BNF in increasing the CYP1 protein level differs with different cell lines. One reason for the variation in the ratio of protein level in both cell lines could be due to CYP1 polymorphisms, especially since these cell lines originate from different individuals. Genetic polymorphisms of CYP1A1 enzymes have been linked with a higher risk for the development of a wide range of cancers (San Jose et al, 2010; Jin et al, 2011). Several studies have also reported the association of CYP1A2 with an increased risk of a wide range of cancers (Landi et al, 1999; Williams et al, 2000). In addition to the above studies, the up-regulation of CYP1B1 has also been shown to be associated with various cancer types (Watanabe, 2000;Ko et al, 2001; Goodman et al, 2001; Tanaka et al, 2002 ; Gatt?s et al, 2006; Varela-Lema, 2008; Ozbek et al, 2010). The results presented in this study provide more evidence about the potential of the CYP1 enzymes as targets in cancer therapy and prevention. 4.4.2. BNF down-regulates the expression of AHR The AHR is a ligand activated basic helix-loop-helix (bHLH) protein that belongs to the Per-Arnt-Sim (PAS) family of transcription factors (Schimidt and Bradfied, 1996). In this study, BNF, which is one of the most potent AHR ligands, was used to investigate the effect it had on the AHR protein expression in esophageal cancer cells, WHCO1 and WHCO6 cells. The results showed a 67 1.3 and 4.5 fold down-regulation of AHR in WHCO1 and WHCO6, respectively, in the presence of the ligand BNF. These results are in agreement with previous studies that showed a down-regulation of AHR protein upon ligand exposure both in vivo and in vitro (Prokipcak and Okey, 1991; Swanson and Perdew, 1993; Giannone et al, 1995; Pollenz, 1996; Giannone et al, 1998; Pollenz et al, 1998; Roman et al, 1998; Davarinos and Pollenz, 1999; Ma and Baldwin, 2000; Wormke et al, 2000; Santiago-Josefat et al, 2001). Previous studies have also shown that the mechanism of AHR downregulation upon ligand binding involves nuclear export of the AHR, ubiquination followed by protein degradation by the 26S proteasome (Pollenz, 1996; Pollenz et al, 1998; Giannone et al, 1998; Pollenz and Barbadour, 2000; Ma and Baldwin, 2000; Pollenz, 2002), which thus cause the reduction of the protein available. This does not occur in control cells, since in control cells, the AHR protein remains inactive and localized in the cytosol where it is not at risk of being ubiquinated and degraded via the 26S proteasome. Another reason for the AHR downregulation in BNF treated cells compared to control cells could involve the AHRR, where the liganded AHR in a heterodimer with ARNT leads to the activation of the AHRR protein, while the up-regulated AHRR, in turn, reduces the expression of AHR (Mimura et al, 1999). 68 4.5. Conclusion In general, the induction of CYP1 enzymes is highly unwanted, since these enzymes have been shown to convert xenobiotics to reactive and carcinogenic metabolites. According to the results obtained, BNF was shown to be a CYP1 inducing compound. BNF may therefore be considered as harmful since exposure to it could lead to an increase in the rate of metabolism of other carcinogenic compounds. The results also showed the differential induction of the CYP1 protein levels after BNF exposure, which suggests the presence of polymorphisms of these enzymes. Together, these results, implicate CYP1 enzymes, especially the CYP1B1, as potential therapeutic targets for cancer prevention. 69 CHAPTER 5: General discussion In South Africa, esophageal cancer is the most prevalent cause of cancer related deaths in black males (Sitas, 1992). Although different factors can play a role in the development of this disease, reports have shown that alcohol consumption and tobacco smoking are the main risk factors for esophageal cancer (Montesano et al, 1996, Castellsaq?e et al, 1999). The development of esophageal cancer is asymptomatic, thus resulting in late diagnosis and poor prognosis. Success in treatment has been limited in terms of esophageal cancer management; therefore, surgery is still the major intervention when the disease is detected relatively early. Therefore, early detection and search for potential anticancer compounds are important in the control of esophageal cancer. Flavonoids, a large class of phenolic compounds present in plants, have been shown to influence a wide range of biological functions, including the inhibition of tumor growth and the prevention of cancer (Harborne et al, 1999). Various natural flavonoids were also shown previously to have antiproliferative activities against several human cancer cell lines (HeLa, MCF7, SK-Mel-28 and KB) (C?rdenas et al, 2006). Considering the antiproliferative activities of some flavonoids on certain cancer cells and due to a lack of such information on esophageal cancer cells, in the current study the antiproliferative activity of a synthetic flavonoid, ?- 70 Naphthofglavone (BNF), on esophageal cancer cells WHCO1 and WHCO6 was evaluated. BNF showed a moderate activity in WHCO6 cells (IC ~ 10?M) and a weak activity in WHCO1 cells (IC50 50 ~ 25?M).Viewed generally, the results show that the growth inhibitory activity of BNF against the two cell lines is not always the same, thus indicating differences in the sensitivity of cancer cells to BNF. The number and position of hydroxyl groups and double bonds are the determining factors for flavonoid antiproliferative activities (Kozikowsk et al, 2003). Flavonoids with 4-6 hydroxyl groups have been shown to have strong antiproliferative activities, whereas those with more or fewer hydroxyl groups show low or no antiproliferative activities (Rice-Evans et al, 1995; Rice-Evans et al , 1996; Rice-Evans, 2001). One of the reasons that BNF did not show the greatest effect on the proliferation of the WHCO1 and WHCO6 cells could be due to its lack of hydroxyl groups. However, the current results still demonstrate that even though the antiproliferative activity of BNF was not outstanding, esophageal cancer cells may still be responsive to the treatment with this flavonoid. Flavonoids contribute to the prevention of cancer through several mechanisms. They may act in different stages of the development of cancer by scavenging free radicals, thus protecting the DNA from oxidative damage (Ferguson, 2001). Flavonoids have been shown to inactivate carcinogens by inhibiting the expression of mutagenic genes. 71 They have also been shown to inactivate enzymes responsible for the activation of procarcinogens and activate the ones responsible for the detoxification of xenobiotics (Bravo et al, 1998). Among the proteins that interact with flavonoids, CYP monooxygenase metabolizing xenobiotics and endogenous substrates play a major role (Hodek et al, 2002). The CYP inhibitory capacity of flavonoids has been widely studied due to their potential use as agents blocking carcinogenesis (Chan et al, 1998; Zhai et al, 1998; Doostdar et al, 2000; Henderson et al, 2000). Certain flavonoids alter the expression of CYP1 enzymes thus inhibiting the activation of procarcinogens to carcinogens and ultimately to a reduction of DNA adduct formation. Reports have shown that flavonoids possessing hydroxyl groups are usually associated with an inhibition of CYP enzyme activity, whereas those lacking hydroxyl groups often stimulate CYP enzyme activity. BNF, which does not possess any hydroxyl groups, and was found to significantly induce the expression of CYP1A1, CYP1A2 and CYP1B1 in both WHCO1 and WHCO6 cells. CYP1A1 is one of the key enzymes in the activation of procarcinogens to carcinogens (Guengerich, 1992; Shimada et al, 1992; Guengerich, 1999; Minsavage et al, 2004; Schwartz et al, 2007). This enzyme was induced by 1.3 and 1.1 fold in WHCO1 and WHCO6 cells, respectively, upon exposure to BNF. The induction of CYP1A1 by BNF may result in an 72 increase in the capacity of CYP1A1 mediated carcinogen activation thus leading to a development of neoplastic diseases. On the other hand, the induction of this enzyme may mean that when exposed to potential carcinogens these carcinogens will be activated and thus be detoxified by phase II enzymes and readily eliminated from the body (Burchell, 2003; Kuuranne et al; 2003). CYP1A2 enzymes are known for their oxidative metabolism of a wide range of therapeutic drugs (Yamazaki et al, 2001; Daniel et al, 2002; Llibre et al, 2002). BNF induced this enzyme by 1.4 fold in both cell lines. Since CYP1A2 metabolizes therapeutic drugs, the simultaneous administration of BNF and therapeutic drugs may cause an altered pharmacokinetics of the drugs thus resulting in either overdose or loss of their therapeutic effect (Tang and Stearns, 2001). Some people are slow drug metabolizers, therefore, the simultaneous administration of their therapeutic drugs and BNF may be beneficial in the activation and improvement of drug transportation in such people. CYP1B1 is well known for the steroid metabolism to the carcinogenic metabolite 4-hydroxyesstradiol (4-OHE2) (Badawi et al, 2001; Cavalieri and Rogan, 2004). This metabolite has been implicated in causing several types of cancer, including breast cancer (Mckay et al, 1995; Liehr and Ricci, 1996; Murray et al, 1997). BNF induced CYP1B1 by 2.4 and 1.7 in 73 WHCO1 and WHCO6 cells, respectively. Induction of CYP1B1 is a risk factor for breast cancer because it is a major enzyme for the carcinogenic estrogen metabolism. A differential expression of the CYP1 proteins was observed in both WHCO1 and WHCO6 cell lines. One reason for the variation in the ratio of protein level in both cell lines could be due to CYP1 polymorphisms that may contribute to interindividual susceptibility to environmental carcinogens. Genetic polymorphisms of CYP1A1 enzymes have been linked with a higher risk for the development of a wide range of cancers. Several studies have also reported the association of CYP1A2 with an increased risk of a wide range of cancers (Landi et al, 1999; Williams et al, 2000). CYP1B1 was highly expressed compared to the CYP1A proteins. This could be because CYP1B1 has been shown to be expressed at high frequencies in various human cancers, including breast, colon, lung, esophagus, skin, lymph node, brain, and testicles but not in normal tissues (Murray et al, 1997; Watanabe, 2000; Ko et al, 2001; Tanaka et al, 2002; Gatt?s et al, 2006; Varela-Lema, 2008). Together, these findings implicate CYP1 enzymes, especially the CYP1B1, as potential therapeutic targets for esophageal cancer prevention. Aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor which regulates CYP1 enzymes. Inhibiting the CYP1 family of enzymes 74 through blocking the AHR plays a critical role in the cancer chemopreventive properties of flavonoids. For instance, quercetin, one of the well known naturally occurring flavonoids, binds as an antagonist to AHR and consequently inhibits benzo(a)pyrene (BP) induced CYP1A1 mRNA transcription and protein expression and thus resulting in decreased BP-DNA adduct formation (Kang et al, 1999). In the present study, BNF did not show an induction but a down-regulation of the AHR in both cell lines. BNF down-regulated AHR by 1.3 and 4.5 fold in WHCO1 and WHCO6, respectively. The mechanism of AHR down- regulation upon ligand binding has been reported to involve nuclear export of the AHR, ubiquination, followed by protein degradation by the 26S proteasome (Pahl and Baeuerle, 1996; Tanaka, 1998; Davarinos and Pollenz, 1999; Ciechanover et al , 2000; Kornitzer and Ciechanover, 2000; Pollenz, 2002). 75 Conclusion and Future studies Summarizing the above results, the agonist function of BNF seems not to be in line with its anticancer properties. However, BNF induction of the CYP1 enzymes may facilitate the removal of other compounds that are already active in the body. This flavonoid may prevent the process of carcinogenesis by other mechanisms such as the induction of both the phase I and phase II enzymes, thus the reactive intermediates that are formed as a result of metabolism of other compounds are removed from the body in a coordinated way. 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Cancer epidemiology biomarkers and prevention 9: 147-50 Zhuo WL, Zhang YS, Wang Y, Zhuo XL, Zhu B, Ca i L, Chen ZT (2009) Association studies of CYP1A1 and GSTM1 polymorphisms with esophageal cancer risk: Evidence-based meta-analyses. Archives of medical research 40:169?179 104 APPENDICES Appendix 1 1.1. MTT cell viability assay 1.1.1. MTT labeling reagent MTT (3-[4, 5-dimethyl thiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) Dissolved in phosphate buffred saline to make a final concentration of 5mg/ml 1.1.2. MTT Solubilization solution 10g SDS Dissolved in 100ML 0.01M HCL 105 Appendix 2 2.1. The effect of DMSO on WHCO 1 cell viability (One-Way anova and Tukey posttest) Table 1.1.B: One-Way ANOVA column statistics for DMSO treated WHCO1 cells Control 0.2 %V/V 0.5%V/V 1%V/V 2.5%V/V 5%V/V Number of values 3 3 3 3 3 3 0.612 0.528 0.512 0.424 0.337 0.067Minimum 0.612 0.528 0.512 0.424 0.337 0.06725% Percentile 0.612 0.548 0.513 0.465 0.346 0.067Median 0.612 0.569 0.526 0.466 0.346 0.0875% Percentile 0.612 0.569 0.526 0.466 0.346 0.08Maximum 0.612 0.5483 0.517 0.4517 0.343 0.07133Mean 0 0.0205 0.00781 0.02397 0.0052 0.00751Std. Deviation 0 0.01184 0.00451 0.01384 0.003 0.00433Std. Error 0.612 0.4974 0.4976 0.3921 0.3301 0.05269Lower 95% CI 0.612 0.5993 0.5364 0.5112 0.3559 0.08998Upper 95% CI 106 Table 1.2: Repeated measures ANOVA for DMSO treated WHCO1 cells P value P<0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 6 F 503.6 R squared 0.996 Was the pairing significantly effective? R squared 0.000000772 F 0.0009757 P value 0.999 P value summary ns Is there significant ma tching? (P < 0.05) No Table 1.3: General ANOVA for DMSO treated WHCO1 cells SS df MS Treatment (between columns) 0.5735 5 0.1147 2.20E- 07 Individual (between rows) 4.44E-07 2 Residual (random) 0.002278 10 0.00023 Total 0.5757 17 107 Table 1.4: Tukey?s multiple comparison test showing the significant difference between the different DMSO concentrations used to treat WHCO1 cells Mean Diff. q Significant? P < 0.05? Summary 95% CI of diff Control vs 0.2 %V/V 0.06367 7.307 Yes ** 0.02087 to 0.1065 Control vs 0.5%V/V 0.095 10 .9 Yes *** 0.05220 to 0.1378 Control vs 1%V/V 0.1603 18 .4 Yes *** 0.1175 to 0.2031 Control vs 2.5%V/V 0.269 30.87 Yes *** 0.2262 to 0.3118 Control vs 5%V/V 0.5407 62.05 Yes *** 0.4979 to 0.5835 0.2 %V/V vs 0.5%V/V 0.03133 3.596 No ns -0.01147 to 0.07413 0.2 %V/V vs 1%V/V 0.09667 11 .09 Yes *** 0.05387 to 0.1395 0.2 %V/V vs 2.5%V/V 0.2053 23.57 Yes *** 0.1625 to 0.2481 0.2 %V/V vs 5%V/V 0.477 54.75 Yes *** 0.4342 to 0.5198 0.5%V/V vs 1%V/V 0.06533 7.498 Yes ** 0.02253 to 0.1081 0.5%V/V vs 2.5%V/V 0.174 19.97 Yes *** 0.1312 to 0.2168 0.5%V/V vs 5%V/V 0.4457 51.15 Yes *** 0.4029 to 0.4885 1%V/V vs 2.5%V/V 0.1087 12.47 Yes *** 0.06587 to 0.1515 1%V/V vs 5%V/V 0.3803 43.65 Yes *** 0.3375 to 0.4231 2.5%V/V vs 5%V/V 0.2717 31.18 Yes *** 0.2289 to 0.3145 108 Appendix 3 3.1 The effect of DMSO on WHCO6 cell viability (One-Way anova and Tukey posttest). Table 1.5: One-way Anova column statistics for DMSO treated WHCO6 cells 0.2 %V/V Control 0.5%V/V 1%V/V 2.5%V/V 5%V/V Number of values 3 3 3 3 3 3 0.4007 0.387 0.355 0.326 0.287 0.158 Minimum 0.4007 0.387 0.355 0.326 0.287 0.158 25% Percentile 0.4007 0.391 0.365 0.334 0.296 0.159 Median 0.4007 0.398 0.377 0.335 0.297 0.166 75% Percentile 0.4007 0.398 0.377 0.335 0.297 0.166 Maximum 0.4007 0.392 0.3657 0.3317 0.2933 0.161 Mean 0 0.005568 0.01102 0.00493 0.005508 0.00436Std. Deviation 0 0.003215 0.00636 0.00285 0.00318 0.00252Std. Error 0.4007 0.3782 0.3383 0.3194 0.2797 0.1502 Lower 95% CI 0.4007 0.4058 0.393 0.3439 0.307 0.1718 Upper 95% CI Table 1.6: Repeated measures ANOVA for DMSO treated WHCO6 cells P value P<0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 6 F 689.2 R squared 0.9971 Was the pairing significantly effective? R squared 0.0008797 F 1.521 P value 0.265 P value summary ns Is there significant ma tching? (P < 0.05) No 109 Table 1.7: General ANOVA for DMSO treated WHCO6 cells SS df MS Treatment (between co lumns) 0.1194 5 0.02388 Individual (between rows) 0.0001054 2 0.00005272 Residual (random) 0.0003466 10 0.00003466 Total 0.1199 17 Table 1.8: Tukey?s multiple comparison test showing the significant difference between the different DMSO concentrations used to treat WHCO6 cells Mean Diff. Significant? P < 0.05? q Summary 95% CI of diff Control vs 0.2 %V/V 0.008667 2.55 No ns -0.008028 to 0.02536 Control vs 0.5%V/V 0.035 10 .3 Yes *** 0.01831 to 0.05170 Control vs 1%V/V 0.069 20.3 Yes *** 0.05231 to 0.08570 Control vs 2.5%V/V 0.1073 31.58 Yes *** 0.09064 to 0.1240 Control vs 5%V/V 0.2397 70 .51 Yes *** 0.2230 to 0.2564 0.2 %V/V vs 0.5%V/V 0.02633 7.748 Yes ** 0.009638 to 0.04303 0.2 %V/V vs 1%V/V 0.06033 17 .75 Yes *** 0.04364 to 0.07703 0.2 %V/V vs 2.5%V/V 0.09867 29.03 Yes *** 0.08197 to 0.1154 0.2 %V/V vs 5%V/V 0.231 67 .96 Yes *** 0.2143 to 0.2477 0.5%V/V vs 1%V/V 0.034 10 Yes *** 0.01730 to 0.05069 0.5%V/V vs 2.5%V/V 0.07233 21 .28 Yes *** 0.05564 to 0.08903 0.5%V/V vs 5%V/V 0.2047 60.22 Yes *** 0.1880 to 0.2214 1%V/V vs 2.5%V/V 0.03833 11 .28 Yes *** 0.02164 to 0.05503 1%V/V vs 5%V/V 0.1707 50 .21 Yes *** 0.1540 to 0.1874 2.5%V/V vs 5%V/V 0.1323 38.94 Yes *** 0.1156 to 0.1490 110 Appendix 4 4.1 The effect of BNF on WHCO1 cell proliferation (One-way anova and Tukey posttest) Table 1.9: One-way ANOVA column statistics for WHCO1 cells treated with BNF for 24 hours Control 5?M 10?M 25?M 50?M 100?M Number of values 3 3 3 3 3 3 1.04 0.9387 0.8637 0.5657 0.5057 0.4557Minimum 1.04 0.9387 0.8637 0.5657 0.5057 0.455725% Percentile 1.04 0.9917 0.9087 0.5667 0.5177 0.4617Median 1.04 0.9917 0.9217 0.5687 0.5207 0.483775% Percentile 1.04 0.9917 0.9217 0.5687 0.5207 0.4837Maximum 1.04 0.974 0.898 0.567 0.5147 0.467Mean 0 0.0306 0.03044 0.00153 0.00794 0.01474Std. Deviation 0 0.01767 0.01757 0.00088 0.00458 0.00851Std. Error 1.04 0.898 0.8224 0.5632 0.4949 0.4304Lower 95% CI 1.04 1.05 0.9736 0.5708 0.5344 0.5036Upper 95% CI 111 Table 1.10: Repeated measures ANOVA for WHCO1 cells treated with BNF for 24 hours P value P<0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 6 F 474.9 R squared 0.9958 Was the pairing significantly effective? R squared 0.0001873 F 0.2233 P value 0.8037 P value summary ns Is there significant ma tching? (P < 0.05) No Table 1.11: General ANOVA for WHCO1 cells treated with BNF for 24 hours SS df MS Treatment (between columns) 0.9752 5 0.195 Individual (between rows) 0.00018 2 9.17E-05 Residual (random) 0.00411 10 0.000411 Total 0.9795 17 112 Table 1.12: Tukey?s multiple comparison test showing the significant difference between the different BNF concentrations used to treat WHCO1 cells after 24 hours Mean Diff. Significant? P < 0.05? q Summary 95% CI of diff Control vs 5M 0.06633 5.669 Yes * 0.008860 to 0.1238 Control vs 10M 0.1423 12.16 Yes *** 0.08486 to 0.1998 Control vs 25M 0.4733 40.45 Yes *** 0.4159 to 0.5308 Control vs 50M 0.5257 44.93 Yes *** 0.4682 to 0.5831 Control vs 100M 0.5733 49 Yes *** 0.5159 to 0.6308 5M vs 10M 0.076 6.495 Yes ** 0.01853 to 0.1335 5M vs 25M 0.407 34.78 Yes *** 0.3495 to 0.4645 5M vs 50M 0.4593 39.26 Yes *** 0.4019 to 0.5168 ?? 5M vs 100M 0.507 43.33 Yes *** 0.4495 to 0.5645 10M vs 25M 0.331 28.29 Yes *** 0.2735 to 0.3885 10M vs 50M 0.3833 32.76 Yes *** 0.3259 to 0.4408 10M vs 100M 0.431 36.84 Yes *** 0.3735 to 0.4885 25M vs 50M 0.05233 4.473 No ns -0.005140 to 0.1098 25M vs 100M 0.1 8.547 Yes ** 0.04253 to 0.1575 50M vs 100M 0.04767 4.074 No ns -0.009807 to 0.1051 113 Table 1.13: One-way ANOVA column statistics for WHCO1 cells treated with BNF for 48 hours 48H BNF Control 5?M 10?M 25?M 50?M 100?M 3 3 3 3 3 3Number of values 1.608 1.469 1.392 0.97 0.9028 0.9503Minimum 1.608 1.469 1.392 0.97 0.9028 0.950325% Percentile 1.608 1.543 1.414 0.971 0.9847 0.974Median 1.608 1.578 1.46 0.9752 0.9954 0.9875% Percentile 1.608 1.578 1.46 0.9752 0.9954 0.98Maximum 1.608 1.53 1.422 0.9721 0.961 0.9681Mean 0 0.05592 0.03508 0.00276 0.05063 0.01572Std. Deviation 0 0.03228 0.02025 0.00159 0.02923 0.00908Std. Error 1.608 1.391 1.335 0.9652 0.8352 0.929Lower 95% CI 1.608 1.669 1.509 0.9789 1.087 1.007Upper 95% CI Table 1.14: Repeated measures ANOVA statistics for WHCO1 cells treated with BNF for 48 hours P value P<0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 6 F 288.9 R squared 0.9931 Was the pairing significantly effective? R squared 0.003087 F 2.252 P value 0.1558 P value summary ns Is there significant ma tching? (P < 0.05) No 114 Table 1.15: General ANOVA statistics for WHCO1 cells treated with BNF for 48 hours SS df MS Treatment (between columns) 1.429 5 0.2858 Individual (between rows) 0.004456 2 0.002228 Residual (random) 0.009895 10 0.0009895 Total 1.443 17 Table 1.16: Tukey?s multiple comparison test showing the significant difference between the different BNF concentrations used to treat WHCO1 cells after 48 hours Mean Diff. q Significant? P < 0.05? Summary 95% CI of diff Control vs 5M 0.07845 4.319 No ns -0.01076 to 0.1677 Control vs 10M 0.1863 10.26 Yes *** 0.09705 to 0.2755 Control vs 25M 0.6363 35.03 Yes *** 0.5471 to 0.7255 Control vs 50M 0.6474 35.65 Yes *** 0.5582 to 0.7366 Control vs 100M 0.6402 35.25 Yes *** 0.5510 to 0.7294 5M vs 10M 0.1078 5.937 Yes * 0.01861 to 0.1970 5M vs 25M 0.5578 30.72 Yes *** 0.4686 to 0.6470 5M vs 50M 0.5689 31.33 Yes *** 0.4797 to 0.6581 5M vs 100M 0.5618 30.93 Yes *** 0.4726 to 0.6510 10M vs 25M 0.45 24.78 Yes *** 0.3608 to 0.5392 10M vs 50M 0.4611 25.39 Yes *** 0.3719 to 0.5503 10M vs 100M 0.454 25 Yes *** 0.3648 to 0.5432 25M vs 50M 0.0111 0.611 No ns -0.07811 to 0.1003 25M vs 100M 0.003957 0.218 No ns -0.08525 to 0.09316 50M vs 100M -0.007144 0.393 No ns -0.09635 to 0.08206 115 Table 1.17: One-way ANOVA column statistics for WHCO1 cells treated with BNF for 72 hours 72H BNF Control 5?M 10?M 25?M 50?M 100?M 3 3 3 3 3 3Number of values 1.668 1.664 1.676 1.264 1.181 1.135Minimum 1.668 1.664 1.676 1.264 1.181 1.13525% Percentile 1.668 1.715 1.676 1.264 1.231 1.161Median 1.668 1.715 1.726 1.278 1.246 1.17375% Percentile 1.668 1.715 1.726 1.278 1.246 1.173Maximum 1.668 1.698 1.692 1.268 1.219 1.156Mean 0 0.02944 0.02887 0.00808 0.03403 0.01943Std. Deviation 0 0.017 0.01667 0.00467 0.01965 0.01122Std. Error 1.668 1.625 1.621 1.248 1.134 1.108Lower 95% CI 1.668 1.771 1.764 1.288 1.304 1.204Upper 95% CI Table 1.18: Repeated measures ANOVA for WHCO1 cells treated with BNF for 72 hours P value P<0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 6 F 327.6 R squared 0.9939 Was the pairing significantly effective? R squared 0.0003594 F 0.2963 P value 0.7499 P value summary ns Is there significant ma tching? (P < 0.05) No 116 Table 1.19: General ANOVA for WHCO1 cells treated with BNF for 72 hours SS df MS Treatment (between columns) 1.021 5 0.2042 Individual (between rows) 0.0003693 2 0.0001847 Residual (random) 0.006233 10 0.0006233 Total 1.028 17 Table 1.20: Tukey?s multiple comparison test showing the significant difference between the different BNF concentrations used treat WHCO1 cells for 72 hours Mean Diff. Significant? P < 0.05? q Summary 95% CI of diff Control vs 5M -0.02967 2.058 No ns -0.1005 to 0.04114 Control vs 10M -0.02433 1.688 No ns -0.09514 to 0.04647 Control vs 25M 0.3997 27.73 Yes *** 0.3289 to 0.4705 Control vs 50M 0.449 31.15 Yes *** 0.3782 to 0.5198 Control vs 100M 0.512 35.52 Yes *** 0.4412 to 0.5828 5M vs 10M 0.005333 0.37 No ns -0.06547 to 0.07614 5M vs 25M 0.4293 29.78 Yes *** 0.3585 to 0.5001 5M vs 50M 0.4787 33.21 Yes *** 0.4079 to 0.5495 5M vs 100M 0.5417 37.58 Yes *** 0.4709 to 0.6125 10M vs 25M 0.424 29.41 Yes *** 0.3532 to 0.4948 10M vs 50M 0.4733 32.84 Yes *** 0.4025 to 0.5441 10M vs 100M 0.5363 37.21 Yes *** 0.4655 to 0.6071 25M vs 50M 0.04933 3.422 No ns -0.02147 to 0.1201 25M vs 100M 0.1123 7.793 Yes ** 0.04153 to 0.1831 50M vs 100M 0.063 4.371 No ns -0.007804 to 0.1338 117 Appendix 5 5.1 The effect of BNF on WHCO6 cell proliferation (One-way anova and Tukey posttest) Table 1.21: One-way ANOVA column statistics for WHCO6 cells treated with BNF for 24 hours Control 5?M 10?M 25?M 50?M 100?M 3 3 3 3 3 3 Number of values 0.7955 0.7305 0.6435 0.5275 0.5015 0.5155 Minimum 0.7955 0.7305 0.6435 0.5275 0.5015 0.5155 25% Percentile 0.7955 0.7375 0.6475 0.5305 0.5045 0.5175 Median 0.7955 0.7425 0.648 0.5415 0.5175 0.5215 75% Percentile 0.7955 0.7425 0.648 0.5415 0.5175 0.5215 Maximum 0.7955 0.7368 0.6463 0.5332 0.5078 0.5182 Mean 0 0.00603 0.00247 0.00737 0.00851 0.00306Std. Deviation 0 0.00348 0.00143 0.00426 0.00491 0.00176Std. Error 0.7955 0.7219 0.6402 0.5149 0.4867 0.5106 Lower 95% CI 0.7955 0.7518 0.6525 0.5515 0.529 0.5258 Upper 95% CI 118 Table 1.22: Repeated measures ANOVA for WHCO6 cells treated with BNF for 24 hours P value P<0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 6 F 1441 R squared 0.9986 Was the pairing significantly effective? R squared 0.00019 F 0.6711 P value 0.5327 P value summary ns Is there significant ma tching? (P < 0.05) No Table 1.23: General ANOVA for WHCO6 cells treated with BNF for 24 hours ANOVA Table SS df MS Treatment (between columns) 0.2267 5 0.04535 Individual (between rows) 4.2E-05 2 2.1E-05 Residual (random) 0.00031 10 3.1E-05 Total 0.2271 17 119 Table 1.24: Tukey?s multiple comparison test showing the significant difference between the different BNF concentrations used to treat WHCO6 cells for 24 hours Mean Diff. q Significant? P < 0.05? Summary 95% CI of diff Control vs 5M 0.05867 18.12 Yes *** 0.04276 to 0.07457 Control vs 10M 0.1492 46.06 Yes *** 0.1333 to 0.1651 Control vs 25M 0.2623 81.01 Yes *** 0.2464 to 0.2782 Control vs 50M 0.2877 88.83 Yes *** 0.2718 to 0.3036 Control vs 100M 0.2773 85.64 Yes *** 0.2614 to 0.2932 5M vs 10M 0.0905 27.95 Yes *** 0.07459 to 0.1064 5M vs 25M 0.2037 62.89 Yes *** 0.1878 to 0.2196 5M vs 50M 0.229 70.71 Yes *** 0.2131 to 0.2449 5M vs 100M 0.2187 67.52 Yes *** 0.2028 to 0.2346 10M vs 25M 0.1132 34.95 Yes *** 0.09726 to 0.1291 10M vs 50M 0.1385 42.77 Yes *** 0.1226 to 0.1544 10M vs 100M 0.1282 39.58 Yes *** 0.1123 to 0.1441 25M vs 50M 0.02533 7.823 Yes ** 0.009426 to 0.04124 25M vs 100M 0.015 4.623 No ns -0.0009070 to 0.03091 50M vs 100M -0.01033 3.191 No ns -0.02624 to 0.005574 120 Table 1.25: One-way ANOVA column statistics for WHCO6 cells treated with BNF for 48 hours Control 5?M 10?M 25?M 50?M 100?M Number of values 3 3 3 3 3 3 Minimum 0.9363 0.8732 0.5039 0.4511 0.4068 0.3836 25% Percentile 0.9363 0.8732 0.5039 0.4511 0.4068 0.3836 Median 0.9363 0.8832 0.5086 0.4562 0.4349 0.3963 75% Percentile 0.9363 0.8845 0.509 0.4613 0.4559 0.3998 Maximum 0.9363 0.8845 0.509 0.4613 0.4559 0.3998 Mean 0.9363 0.8803 0.5072 0.4562 0.4325 0.3932 Std. Deviation 0 0.00617 0.00285 0.00511 0.02463 0.0085 Std. Error 0 0.00357 0.00165 0.00295 0.01422 0.00491 Lower 95% CI 0.9363 0.865 0.5001 0.4435 0.3713 0.3721 Upper 95% CI 0.9363 0.8956 0.5143 0.4689 0.4937 0.4143 Table 1.26: Repeated measures ANOVA for WHCO6 cells treated with BNF for 48 hours P value P<0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 6 F 1617 R squared 0.9988 Was the pairing significantly effective? R squared 0.000479 F 1.938 P value 0.1945 P value summary ns Is there significant ma tching? (P < 0.05) No 121 Table 1.27: General ANOVA for WHCO6 cells treated with BNF for 48 hours SS df MS Treatment (between columns) 0.8754 5 0.1751 Individual (between rows) 0.00042 2 0.00021 Residual (random) 0.00108 10 0.00011 Total 0.8769 17 Table 1.28: Tukey?s multiple comparison test showing the significant difference between the different BNF concentrations used to treat WHCO6 cells for 48 hours Mean Diff. q Significant? P < 0.05? Summary 95% CI of diff Control vs 5M 0.05603 9.326 Yes *** 0.02652 to 0.08554 Control vs 10M 0.4292 71.43 Yes *** 0.3996 to 0.4587 Control vs 25M 0.4802 79.92 Yes *** 0.4506 to 0.5097 Control vs 50M 0.5038 83.86 Yes *** 0.4743 to 0.5333 Control vs 100M 0.5431 90.39 Yes *** 0.5136 to 0.5726 5M vs 10M 0.3731 62.1 Yes *** 0.3436 to 0.4026 5M vs 25M 0.4241 70.59 Yes *** 0.3946 to 0.4536 5M vs 50M 0.4478 74.53 Yes *** 0.4183 to 0.4773 5M vs 100M 0.4871 81.07 Yes *** 0.4576 to 0.5166 10M vs 25M 0.05099 8.487 Yes ** 0.02148 to 0.08050 10M vs 50M 0.07466 12.43 Yes *** 0.04515 to 0.1042 10M vs 100M 0.1139 18.96 Yes *** 0.08443 to 0.1435 25M vs 50M 0.02367 3.94 No ns -0.005842 to 0.05318 25M vs 100M 0.06295 10.48 Yes *** 0.03344 to 0.09247 50M vs 100M 0.03928 6.538 Yes ** 0.009772 to 0.06880 122 Table 1.29: One-way ANOVA column statistics for WHCO6 cells treated with BNF for 72 hours Contro l 72h BNF 5?M 10?M 25?M 50?M 100?M Number of values 3 3 3 3 3 3 Minimum 1.135 1 0.637 0.423 0.375 0.314 25% Percentile 1.135 1 0.637 0.423 0.375 0.314 Median 1.135 1.025 0.688 0.446 0.377 0.332 75% Percentile 1.135 1.035 0.688 0.478 0.394 0.34 Maximum 1.135 1.035 0.688 0.478 0.394 0.34 Mean 1.135 1.02 0.671 0.449 0.382 0.3287 Std. Deviation 0 0.01803 0.02944 0.02762 0.01044 0.01332 0.00602 8 0.00768 8 Std. Error 0 0.01041 0.017 0.01595 Lower 95% CI 1.135 0.9752 0.5979 0.3804 0.3561 0.2956 Upper 95% CI 1.135 1.065 0.7441 0.5176 0.4079 0.3617 Table 1.30: Repeated measures ANOVA for WHCO6 cells treated with BNF for 72 hours P value P<0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 6 F 945.8 R squared 0.9979 Was the pairing significantly effective? R squared 0.000432 F 1.024 P value 0.3939 P value summary ns Is there significant ma tching? (P < 0.05) No 123 Table 1.31: General ANOVA for WHCO6 cells treated with BNF for 72 hours SS df MS Treatment (between co lumns) 1.76 5 0.3519 Individual (between rows) 0.000762 2 0.000381 Residual (random) 0.003721 10 0.000372 Total 1.764 17 Table 1.32: Tukey?s multiple comparison test showing the significant difference between the different BNF concentrations used to treat WHCO6 cells for 72 hours Tukey's Multiple Comparison Test Significant? P < 0.05? Mean Diff. q Summary 95% CI of diff Control vs 5M 0.1147 10.3 Yes *** 0.05997 to 0.1694 Control vs 10M 0.4637 41.64 Yes *** 0.4090 to 0.5184 Control vs 25M 0.6857 61.57 Yes *** 0.6310 to 0.7404 Control vs 50M 0.7527 67.59 Yes *** 0.6980 to 0.8074 Control vs 100M 0.806 72.38 Yes *** 0.7513 to 0.8607 5M vs 10M 0.349 31.34 Yes *** 0.2943 to 0.4037 5M vs 25M 0.571 51.27 Yes *** 0.5163 to 0.6257 5M vs 50M 0.638 57.29 Yes *** 0.5833 to 0.6927 5M vs 100M 0.6913 62.08 Yes *** 0.6366 to 0.7460 10M vs 25M 0.222 19.93 Yes *** 0.1673 to 0.2767 10M vs 50M 0.289 25.95 Yes *** 0.2343 to 0.3437 10M vs 100M 0.3423 30.74 Yes *** 0.2876 to 0.3970 25M vs 50M 0.067 6.016 Yes * 0.01230 to 0.1217 25M vs 100M 0.1203 10.81 Yes *** 0.06563 to 0.1750 50M vs 100M 0.05333 4.789 No ns -0.001368 to 0.1080 124 Appendix 6 6.1 The median inhibition concentration (IC ) values 50 Table 1.33: The median inhibition concentration (IC50) values of BNF in WHCO1 cells BNF CONC Absorbance values Percentage Average IC STDEV 50 0?M 1.040334 1.040334 1.040334 100 100 100 100 0 5?M 0.938667 0.991667 0.991667 90.22747 95.32198 95.32198 93.62381 2.941321 10?M 0.921667 0.863667 0.908667 88.59337 83.01824 87.34378 86.31846 2.925572 25?M 0.566667 0.568667 0.565667 54.46972 54.66196 54.3736 54.50176 ~ 25?M 0.14683 50?M 0.505667 0.520667 0.517667 48.60622 50.04806 49.75969 49.47132 0.762952 100?M 0.461667 0.483667 0.455667 44.37681 46.49151 43.80007 45.43416 1.417067 Table 1.34: The median inhibition concentration (IC50) values of BNF in WHCO6 cells BNF CONC Absorbance values Percentage Average IC STDEV 50 0?M 0.936333 0.936333 0.936333 0.93633 100 100 100 100 100 0 5?M 0.884473 0.873209 0.883224 94.46137 93.2584 94.328 94.01591 0.659422 10?M ~10?M 0.508988 0.503887 0.489733 0.50864 54.35972 53.8149 52.3033 54.3231 53.70025 0.9639 25?M 0.451072 0.456183 0.461294 48.17431 48.7202 49.266 48.72017 0.545853 50?M 0.45609 0.406796 0.434851 0.45589 48.71023 43.4457 46.4419 48.6891 46.82172 2.489687 100?M 0.430515 0.383627 0.396276 0.39978 45.97883 40.9712 42.3221 42.6967 42.99221 2.124516 Note: The one-way anova results for the other compounds are not shown. 125 Appendix 7 7.1. Protein extraction Laemmli samp le buffer 1.5% Tris.HCL (pH 6.8) 2% SDS 10% Glycerol 5% ?-mercaptoethanol Make up in dH 2 O Store at 4?C 7.2 Protein estimation Trichloroacetic acid (TCA) 7.5% TCA Make up to the requ ired volume with dH 2 O Coomassie Blue solution (0.25%) 0.25% Coomassie brilli ant blue powder 50% Methanol 10% Glacial acetic acid Make up to final volume with dH2O Destaining solution 12% Glacial acetic acid 10% Methanol Make up to final volume with dH2O Elution solution 66% Methanol 33% dH2O 1% Ammonia 126 Estimation of th e total protein content of cells Protein Estimation Standard currve y = 0.0102x + 0.013 R2 = 0.9965 0 0.05 0.1 0.15 0.2 0.25 0 5 10 15 20 25 BSA(ug) A b so rb an ce ( 59 6 n m ) Figure 7.1: BSA standard curve for protein estimation. Shown is a plot of the absorbance values at 596nm versus BSA concentrations (1-20?g). The equation of the standard curve(y=0.0102x + 0.013) was used to calculate the unknown concentrations of the whole cell extracts. R2 (Linear regression) =0.9965. 7.3 SDS-PAGE Separating gel 10% Acrylamide 0.1% NN?-methylenebisacrylamide 375 mM Tris-HCl, pH 8.8 0.2% SDS Make up to final volume with dH2O Just before use add: 1mM Ammonium persulphate 0.25% N, N, N?N?-tetramethylenediamine (TEMED) 127 Stacking gel 10% Acrylamide 0.1% NN?-methylenebisacrylamide 125 mM Tris-HCl, pH 6.8 0.2% SDS Make up to final volume with dH2O Just before use add: 1mM Ammonium persulphate 0.25% TEMED Running buffer 3.74 mM SDS 25 mM Tris-HCl, pH 8.3 192.5 mM Glycine Make up to final volume with dH2O Destain solution 10% Acetic acid 10% Methanol Make up to final volume with dH2O 7.4 Western Blot Transfer buffer 25 mM Tris-HCl, pH 8.3 1.41% Glycine 20% Methanol Make up to final volume wi th dH2O (Store at4?c) Blocking solution 50 mM Tris-HCl, pH 7.8 2 mM Calcium chloride dihydrate 5% non-fat milk powder 0.01% anti-foam 0.05% Triton-X 100 Make up to final volume with dH2O Store at 4?C 128 Super signal west pico chemiluminescent substrate kit Before use mix : 50% Luminol/Enhancer solution 50% Stable peroxide buffer Store in the dark Developer 6.4 M Metol 0.6 M Sodium sulphite (anhydrous) 80 mM Hydroquinine 0.45 mM Sodium carbonate (anhydrous) 34 mM Potassium bromide Make up to final volume with dH2O Store in the dark Fixer 0.8 M Sodium trisulphate 0.2 M Sodium metasulphite Make up to final volume with dH2O Store in the dark 129 Appendix 8 8.1 T-test results for comparin g protein expression level Table 1.35: CYP1A1 fold induction WHCO1 BNF treatment WHCO6 BNF treatment Fold induction 1.348312 1.327196 1.098469 1.075601 1.075601 Fold induction average 1.337754 1.083224 STDEV 0.014931 0.013203 P 0.019894 0.008285 Table 1.36:CYP1A2 fold induction WHCO1 BNF treatment WHCO6 BNF treatment Fold induction 1.352497 1.36784 1.476955 1.399223 Fold induction average 1.360169 1.438089 STDEV 0.010849 0.054965 P 0.013558 0.056332 130 Table 1.37:CYP1B1 fold induction WHCO1 BNF treatment WHCO6 BNF treatment Fold induction 2.467483 2.276516 2.517369 1.676021 1.794708 Fold induction average 2.420456 1.735364 STDEV 0.127127 0.083925 P 0.002659 0.051264 Table 1.38: AHR fold induction WHCO1 BNF treatment WHCO6 BNF treatment Fold induction 0.751155 0.722407 0.768886 0.22846 0.218438 Fold induction average 0.747483 0.223449 STDEV 0.023456 0.007087 P 0.002864 0.004108 131