UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Effects of short-term supplementation of folic acid and L-5-methyltetrahydrofolate on cell proliferation… Hempstock, Wendy 2014

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2014_spring_hempstock_wendy.pdf [ 2.21MB ]
Metadata
JSON: 24-1.0167325.json
JSON-LD: 24-1.0167325-ld.json
RDF/XML (Pretty): 24-1.0167325-rdf.xml
RDF/JSON: 24-1.0167325-rdf.json
Turtle: 24-1.0167325-turtle.txt
N-Triples: 24-1.0167325-rdf-ntriples.txt
Original Record: 24-1.0167325-source.json
Full Text
24-1.0167325-fulltext.txt
Citation
24-1.0167325.ris

Full Text

       Effects of short-term supplementation of folic acid and L-5-methyltetrahydofolate on cell proliferation and the expression of folate transporters in human colorectal adenocarcinoma (Caco2) cells  by Wendy Hempstock B.Sc., The University of British Columbia, 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Human Nutrition)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2014  © Wendy Hempstock, 2014    ii  Abstract Folate plays a role in the synthesis and repair of DNA and the generation of methyl groups. Folic acid (FA) is a synthetic oxidized form of folate used in food fortification and supplements in Canada. Increased colon cancer incidence has been correlated with FA fortification in several countries. The effect of FA on the development of colon cancer is controversial as other research shows a lack of association between FA fortification and colon cancer incidence.  I hypothesize that FA affects proliferation and folate transporter expression in colon cancer cells differently than L-5-methyltetrahydrofolate (5MTHF). In addition, the forms of folate, reduced versus oxidized, would differentially affect the activity of the Wnt signaling pathway. The overall objective of my research is to investigate the effect of FA and 5MTHF on cell proliferation, the expression of selected folate transporters, and the activity of the Wnt signalling pathway in human colorectal adenocarcinoma (Caco2) cells. Caco2 cells were cultured for 3 or 5 days in folate-free RPMI 1640 medium supplemented with 10% dialyzed FBS and treated with 0, 0.9, 2.3, or 3.4 µM FA or MTHF. Cell viability was assessed using WST-1 colourimetric assay. Cell proliferation was assessed by BrdU colourimetric assay and cell cycle analysis with BrdU incorporation was measured by flow cytometry. The abundance of reduced folate transporter (RFC), folate receptor-α (FRα), proton-coupled folate transporter (PCFT), breast cancer resistance protein (BCRP) was assessed by Western blotting. β-Catenin nuclear localization was assessed by measuring the fluorescence of Alexa Fluor 488® using confocal microscopy. FA treatment increased cell proliferation compared to treatment with MTHF at all concentrations after 3 days. After 5 days, there was no difference in cell viability or cell iii  proliferation. Cell cycle analysis after 5 days of 3.4 µM FA and 5MTHF treatment showed spikes in the pre-G1 phase compared to the control. Neither folate transporter expression nor β-Catenin nuclear localization was affected by FA and 5MTHF treatment under the conditions tested. This lack of effect of FA and 5MTHF on cell proliferation and the expression of selected folate transporters was possibly due to relatively short treatment duration.                  iv  Preface  This graduate thesis was prepared in accordance to the University of British Columbia Faculty or Graduate Studies requirements. I was responsible for performing all experiments. The research design, interpretation of the results, and preparation of this thesis were accompanied with the assistance and guidance of Dr. Zhaoming Xu.                                     v  Table of Contents  Abstract  ............................................................................................................................. ii Preface   ............................................................................................................................. iv Table of Contents  ..............................................................................................................v List of Tables  ................................................................................................................. viii List of Figures  .................................................................................................................. ix List of Abbreviations  ........................................................................................................x Acknowledgements   ....................................................................................................... xii Introduction  .......................................................................................................................1 Chapter 1 Literature Review, Hypothesis, and Objectives  ...........................................3 1.1 Folate  ..........................................................................................................3 1.1.1 Dietary Sources  ................................................................................3 1.1.2 Recommended Daily Allowance (RDA) and Folate  .......................3 1.1.3 Digestion and Absorption  ................................................................6 1.1.4 Physiological Functions  .................................................................11 1.1.5 Interactions with Vitamin B12  ........................................................12 1.1.6 Deficiency  ......................................................................................13 1.1.7 Neural Tube Defects and Folic Acid Fortification  ........................16 1.2 Wnt Signalling Pathway ...........................................................................18 1.2.1 Overview  ........................................................................................18 1.2.2 β-Catenin-dependent Wnt Signalling Pathway  ..............................19 1.2.3 β-Catenin-independent Wnt Signalling Pathway  ...........................22 1.3 Linking Folic Acid and Wnt Signalling to Colorectal Cancer  .................24 1.3.1 Folic Acid and Colorectal Cancer  ..................................................24          1.3.1.1 Protective Effects of Folate Intakes against Cancer  ..........24          1.3.1.2 Promoting Effects of Folate Intake on Cancer                      Development  ......................................................................26          1.3.1.3 Possible Mechanisms of Folate’s Role in Colorectal                       Cancer  ................................................................................30   1.3.2 Wnt Signalling and Colorectal Cancer  ..........................................33 1.3.3 Evidence for Wnt and Folate Interactions in Colorectal Cancer  ...36 1.4 Summary  ..................................................................................................42 1.5 Hypothesis  ................................................................................................43 1.6 Overall Objective and Specific Aims  .......................................................43 vi  Chapter 2 Effects of Short-term Supplementation of Folic Acid and L-5-                    methyltetrahydofolate on Proliferation, Viability and the Expression                    of Folate Transporters in Human Colorectal Adenocarcinoma (Caco2)                   Cells  ................................................................................................................50 2.1 Introduction  ..............................................................................................50 2.2 Material and Methods  ..............................................................................52 2.2.1 Cell Culture System and Folate Treatments ...................................52 2.2.2 Assessment of Cell Viability and Proliferation  .............................53 2.2.3 Cell Cycle Analysis.........................................................................54 2.2.4 Whole Cell Lysate Preparation and Western Blot Analysis  ..........57 2.2.5 Cellular Localization of β-Catenin  ................................................59 2.2.6 Statistical analysis  ..........................................................................60 2.3 Results  ......................................................................................................61 2.3.1 Cell Viability  ..................................................................................61 2.3.2 Cell Proliferation  ............................................................................61 2.3.3 Cell Cycle Analysis ........................................................................62 2.3.4 Folate Transporters  ........................................................................63 2.3.5 Nuclear Localization of β-Catenin  .................................................63 2.4 Discussion  ................................................................................................64 2.4.1 Folate Treatment Did Not Affect Caco2 Cell Viability and          Proliferation  ...................................................................................64 2.4.2 Apparently Differential Effect of FA and 5MTHF on Cell   Viability and Proliferation  .............................................................67 2.4.3 The Effect of Folate Treatment on the Abundance of Folate          Transporters  ...................................................................................74 2.4.4 Nuclear Localization of β-Catenin was Unaffected by Folate          Treatment  .......................................................................................78 2.4.5 Summary  ........................................................................................79 Chapter 3 Limitations and Future Directions  ..............................................................93 3.1 Limitations  ...............................................................................................93 3.2 Future directions  ......................................................................................95       3.2.1 Treatment length  .............................................................................96       3.2.2 Cell type  ..........................................................................................96       3.2.3 Gene expression  ..............................................................................97 References  ........................................................................................................................99 vii  Appendices  .....................................................................................................................117 Appendix A: Flow Cytometry Diagrams  .....................................................117 Appendix B: Raw Data for β-Catenin Nuclear Localization  .......................123                                           viii  List of Tables Table 1.1: Food Sources of Folate  ....................................................................................45 Table 2.1: Antibodies Used in the Western Blots  .............................................................80 Table 2.2: Cell Cycle Analysis of Caco2 Cells Following 3 Day Treatment with FA                  5MTHF  ............................................................................................................81 Table 2.3: Cell Cycle Analysis of Caco2 Cells Following 5 Day Treatment with FA                  5MTHF  ............................................................................................................82 Table B.1:Raw Data for β-Catenin Nuclear Localization  ...............................................123                   ix  List of Figures Figure 1.1: The Structure of Folic Acid and 5-methyltetrahydrofolate  ............................46 Figure 1.2: The Folate Metabolism Pathway  ....................................................................47 Figure 1.3: Schematic of Canonical Wnt/β-Catenin-dependent Wnt Signalling  ..............48 Figure 1.4: Colorectal Cancer Rates Pre- and Post-fortification in the U.S. Canada  .......49 Figure 2.1: Cell Viability of Caco2 Cells after 3 Day Treatment with FA or 5MTHF  ....83 Figure 2.2: Cell Viability of Caco2 Cells after 5 Day Treatment with FA or 5MTHF  ....84 Figure 2.3: Cell Proliferation of Caco2 Cells after 3 Day Treatment with FA or                     5MTHF  ..........................................................................................................85 Figure 2.4: Cell Proliferation of Caco2 Cells after 5 Day Treatment with FA or                     5MTHF  ..........................................................................................................86 Figure 2.5: Relative Protein Abundance of RFC in Caco2 Cells after 5 Day Treatment                    with FA or 5MTHF  ........................................................................................87 Figure 2.6: Relative Protein Abundance of FRα in Caco2 Cells after 5 Day Treatment                    with FA or 5MTHF  ........................................................................................88 Figure 2.7: Relative Protein Abundance of BCRP in Caco2 Cells after 5 Day Treatment                    with FA or 5MTHF  ........................................................................................89 Figure 2.8: β-Catenin Fluorescence (Ratio of Nucleus to Cytoplasm) in Caco2 Cells                      after 5 days treatment with FA or 5MTHF  ...................................................90 Figure 2.9: β-Catenin Fluorescence in Caco2 Cells after 5 Days Treatment with FA or                      5MTHF  ........................................................................................................91 Figure A.1: Cell Cycle Analysis of Caco2 Cells after 3 Day Treatment with FA or                    5MTHF  ........................................................................................................117 Figure A.2: Cell Cycle Analysis of Caco2 Cells after 5 Day Treatment with FA or                    5MTHF .........................................................................................................120        x  List of Abbreviations 5MTHF                                                                                          5-methyltetrahydrofolate ABC                                                                                         ATP binding cassette protein AICAR                                  Phosphoribosylaminoimidazolecarboxamide transformylase ANOVA                                                                                                Analysis of variance AOM                    Azoxymethane APC                                                                                          Adenomatous polyposis coli ATP                                                                                                  Adenosine triphosphate BCRP                                                                                  Breast cancer resistance protein BrdU                                                                                            5-Bromo-2’-deoxyuridine BSA                                                                                                   Bovine serum albumin CamKII                                                                Calcium/calmodulin-dependent kinase II CI                                                                                                           Confidence interval CK1                                                                                                              Casein kinase 1 CNS                                                                                                  Central nervous system CRD                                                                                                     Cysteine rich domain Daam1                                               Dishevelled associated activator of morphogenesis 1 DAPI                                                                                    4’,6-diamidino-2-phenylindole dFBS                                                                                         Dialyzed fetal bovine serum DFE                                                                                              Dietary folate equivalents DRI                                                                                                  Dietary reference intake DSH                                                                                                        Dishevelled protein dTMP                                                                               Deoxythymidine monophosphate dUMP                                                                                    Deoxyuridine monophosphate DHF                                                                                                                 Dihydrofolate DKK                                                                                                           Dickkopf protein DMEM                                                                    Dulbecco’s modified essential medium DNA                                                                                                   Deoxyribonucleic acid EAR                                                                                     Estimated average requirement ER                                                                                                     Endoplasmic reticulum EDTA                                                                                 Ethylenediaminetetraacetic acid FA                                                                                                                          Folic acid FAP                                                                                    Familial adenomatous polyposis FBS                                                                                                          Fetal bovine serum FR                                                                                                                  Folate receptor FZD                                                                                                              Frizzled protein GAR                                                                 Phosphoribosylglycinamide transformylase GPI                                                                                        Glycosyl phosphatidylinositol GSK3                                                                                         Glycogen synthase kinase 3 HCl                                                                                                           Hydrochloric acid HCP1                                                                                                 Heme carrier protein 1 HFM                                                                                   Hereditary folate malabsorption HR                                                                                                                      Hazard ratio ISAM                                                                       Intestinal surface acidic microclimates LRP5/6                                                                  Lipoprotein receptor-related protein 5/6 xi  mRNA                                                                                                         Messenger RNA MRP                                                                                         Multidrug resistance protein MTHFR                                                                      Methylenetetrahydrofolate reductase NTD                                                                                                       Neural Tube Defect PBS                                                                                               Phosphate buffered saline PBT                                                                                              PBS containing Tween-20 PBTB                                                                           PBS containing Tween-20 and BSA PBTG                                                                          PBTB containing normal goat serum PCFT                                                                                Proton-coupled folate transporter PCP                                                                                                         Planar cell polarity PDE                                                                                                          Phosphodiesterase  PI                                                                                                                Propidium iodide PKC                                                                                                             Protein kinase C PLC                                                                                                             Phospholipase C PMSF                                                                                 Phenylmethylsulphonyl-fluoride Porc                                                                                                           Porcupine protein PVDF                                                                                               Polyvinylidene fluoride RBC                                                                                                                Red blood cell RDA                                                                                     Recommended daily allowance RFC                                                                                                    Reduced folate carrier ROCK                                                                                                Rho associated kinase RNA                                                                                                            Ribonucleic acid RNase                                                                                                             Ribonucleases RR                                                                                                                         Risk Ratio SAH                                                                                             S-Adenosylhomocysteine SAM                                                                                                 S-Adenosylmethionine SDS                                                                                                Sodium dodecyl sulphate SFRP                                                                                  Soluble frizzled-related proteins SHMT                                                                               Serine hydroxymethyltransferase siRNA                                                                                                Small interfering RNA TBS                                                                                                        Tris-buffered Saline TCF/LEF                                                  T cell factor/Lymphoid enhancer-binding factor TTBS                                                                                           Tris-tween buffered saline THF                                                                                                            Tetrahydrofolate VEGF                                                                             Vascular endothelial growth factor Wg                                                                                                              Wingless protein WIF                                                                                                    Wnt inhibitory protein Wls                                                                                        Wntless/Wnt sorting receptors Wnt                                                                                                     Wingless type protein WST-1   4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate    xii  Acknowledgements My graduate study has been a hard-fought and long-lasting battle. I would like to thank everyone at the University of British Columbia who has helped me throughout the process of completing my Master’s degree. Especially, I would like to acknowledge and thank my supervisor, Dr. Zhaoming Xu, for his ongoing patience, encouragement, support and guidance throughout my research and the completion of this thesis. Without him, I may not have been able to finish the final steps of my graduation requirements. I would also like to thank my supervisory committee members, Dr. Angela Devlin and Dr. David Kitts, for their expertise and valuable insights into the research project and my external examiner, Dr. Christine Scaman, for her involvement.  This work would not have been possible without funding support from AFMnet and the UBC Vitamin and Mineral Research Fund.  Thank you also to my lab mates, Deanna Ibbitson, Alice Lin, Melinda Bakker, and Li He, for their support and friendship during this time. I would also like to thank Helen Chan and Jon Lee for their help and support in the lab. Lastly, special thanks to my beloved family and friends for their unconditional love, encouragement and support.  1  Introduction Folate is a generic term for the water-soluble B-vitamin that plays an important role in DNA synthesis and repair, methylation, and amino acid metabolism. Folate deficiency in humans is characterized by impaired DNA synthesis, which disrupts cell division, and presents clinically as megaloblastic anemia (Vilter et al., 1963; Herbert, 1964).  Folate supplementation has been associated with a decreased incidence of neural tube defects (NTDs; MRC Vitamin Study Research Group, 1991). Higher folate intakes have been suggested as protective against certain types of cancer, especially colorectal (Blount et al., 1997; Giovannucci et al., 1998; Sanjoaquin et al., 2005; Kim et al., 2010; Lee et al. 2011). The relationship between folate intake and colorectal cancer is controversial as there are studies correlating the start of fortification of folic acid (FA), the oxidized form of folate, with an increase in colorectal cancer rates and studies pointing to over-supplementation of FA contributing to the development of cancer (Mason et al., 2007; Ebbing et al., 2009; Figueiredo et al., 2009; Hirsch et al., 2009; Nyström and Mutanen, 2009). The reasons for the apparent contradictory effects of FA intake and cancer are as yet unknown, although it is thought that the timing of folate exposure or supplementation is important to determine whether folate will be associated with an increased or decreased risk of cancer (Ulrich and Potter, 2007). Since 1998, flour and grain products such as cereal, pasta and cornmeal have been fortified with FA to prevent folate-related NTDs in Canada and the United States. At the time it was also theorized that FA fortification could have beneficial effects on cardiac events (by lowering homocysteine) and cancer incidence.  In addition to FA fortification, Health Canada also recommended that all women of childbearing age to take a daily 2  supplement of 400 μg FA.  FA is used in food fortification and vitamin supplements due to its chemical stability against oxidation and therefore is used in food products requiring cooking or long term storage.  However, FA is a synthetic form of the vitamin and is not biologically active. Presently, it is not known whether this synthetic form of folate has different effects on cancer cell growth compared to the naturally occurring form of folate.  The Wnt signalling pathway is constitutively active in most colon cancers. It is so prevalent that the next generation of colon cancer treatments will be aimed at preventing or controlling Wnt signalling to stop cancer progression. Loss of regulation of Wnt signalling is likely the starting point for many colon cancers. Since this pathway is also aberrant in NTDs (some of which are folate-sensitive), a connection between Wnt signalling and folate needs to be investigated.                         3  CHAPTER 1 Literature Review, Hypothesis, and Objectives   1.1  Folate   1.1.1 Dietary Sources Folate is a water-soluble B vitamin.  Folate is a term that refers to reduced forms of the vitamin that occur naturally in foods and tissues in humans (Figure 1.1).  FA is a synthetic, oxidized derivate of the vitamin that is used in supplements and in fortification of flour, pasta and cereals (Figure 1.1). Dietary sources of folate (Table 1.1) are leafy green vegetables (e.g. spinach, broccoli, okra and asparagus), legumes (e.g. black, kidney, navy and pinto beans, chick peas and lentils), fruit (e.g. oranges) and organ meat (Cuskelly et al., 1996; West Suitor and Bailey, 2000). Folate is present mostly as dihydrofolate (DHF) in foods of plant origin and as 5-methyltetrahydrofolate (5MTHF) in foods of animal origin. Naturally occurring folates have a polyglutamate tail.  In Canada, flour, pasta, and cereals are fortified with FA due to its chemical stability.  FA has also been used in supplements. Because of its monoglutamate tail, FA is more readily absorbed than naturally occurring folates and therefore is considered to have a higher bioavailability.   1.1.2 Recommended Daily Allowance (RDA) and Folate Intake in Canada   Dietary folate equivalents (DFE) are used to account for the differences of bioavailability between food folates and FA (West Suitor and Bailey, 2000). A DFE is defined as 1 g of naturally occurring food folate, which is equal to 0.6 g of FA from a 4  supplement or fortified food consumed with a meal, or 0.5 g of FA from a supplement consumed on an empty stomach (DRI, 1998).   The bioavailability of natural food folates appears to be 50% of that of FA, based on a study in which 10 non-pregnant, healthy women were monitored in a metabolic unit for 92 days and the ability of naturally occurring food folates or FA to raise or maintain plasma folate levels after a 28 day folate depletion period was examined (Sauberlich et al., 1987). Another study involving a 4-week dietary intervention, determined that the bioavailability of food folates (from fruit, vegetables, and liver) is 80% of that of FA after comparing 4 diets supplemented with varying levels of [13C11]-labelled FA (Winkels et al., 2007). Actually, true bioavailability of folates cannot be determined because of modifications such as formylation or methylation that occur in the intestinal mucosa after absorption, so the bioavailability of the folate relative to the bioavailability of the fully oxidized monoglutamate form of the vitamin (FA) is determined (Brouwer et al., 2001). Evidently, FA is more biologically available than folate. The RDA of folate for Canadians aged 14 and above is 400 μg DFE/day, for pregnant women it is 600 μg DFE/day, and for lactating women it is 500 μg DFE/day (Health Canada DRI reference tables, 2010). In addition, Health Canada also recommends that women of childbearing age take a supplement containing 400 μg FA daily (Health Canada, 2010).  The Canadian Community Health Survey cycle 2.2 (CCHS 2.2) reported that in some groups of Canadians, over 10% of people had an inadequate intake of folate. Over 10% of men ( 51 y) and women ( 14 y) had inadequate folate intake (CCHS 2.2, 2004). The worst inadequacies are found in women over the age of 14 and men aged 71 and 5  over, whose inadequate folate intakes reached an incidence of more than 20% (CCHS 2.2, 2004). It is estimated that the number of British Columbians with folate intakes less than the estimated average requirement (EAR) is lower than the national average (15.5% versus 15.7%; CCHS 2.2, 2004). Even with mandatory FA fortification of flours, cereals, pasta and cornmeal, there are still groups of Canadians that are not meeting the EAR for folate intake. This is important in the case of women of childbearing age (approximately 20% of women aged 14 – 50 years do not meet the requirements of the EAR for folate; CCHS 2.2, 2004), the original target of FA fortification. A survey of women of child-bearing age (aged 18 – 45 years) from Vancouver, Canada showed that 86% meet the EAR for folate, but only 26% of women met the recommended levels of folate for women who are capable of getting pregnant (French et al., 2003). Another study focusing on Canadian women (aged 18 – 25 years), showed that only 17% of women met the recommended levels of folate for women capable of getting pregnant (Shuaibi et al., 2008). Analysis of the Canadian Health Measures Survey shows that less than 1% of Canadians have folate deficiency according to their red blood cell folate and more than 40% showed high red blood cell folate (>1360 nmol/L; Colapinto et al., 2011). Looking at women of childbearing age, 22% do not meet the red blood cell folate recommendations for maximal reduction of risk of NTDs (<906 nmol/L; Colapinto et al., 2011). Women of childbearing age were the original targets of the folate fortification program.   6  1.1.3 Digestion and Absorption Folate is absorbed in monoglutamate form (Reisenauer et al., 1977). Naturally occurring folates have a polyglutamate tail that is cleaved by glutamate carboxypeptidase II before absorption (Reisenauer et al., 1977). FA does not require glutamate carboxypeptidase II-mediated cleavage before absorption because it is in the monoglutamate form. The lack of a polyglutamate tail makes FA more bioavailable than naturally occurring folates.  Proton coupled folate transporter (PCFT).  Folates are currently thought to be absorbed mainly in the duodenum and proximal jejunum via PCFT, a membrane-bound transport protein. PCFT was initially identified as heme carrier protein 1 (HCP-1; SLC46A1; Qiu et al., 2006). PCFT is expressed at the highest levels in the duodenum and jejunum, and lower levels in the ileum and colon.  It exhibits greatest folate transport activity at pH 5.5 (Qiu et al., 2006).  PCFT exerts its function in the intestine in humans and rats because of the presence of intestinal surface acidic microclimates (ISAM; Lucas and Blair, 1978; Said et al., 1987). The pH of ISAM has been found to increase from the duodenum to the ileum with further increases in the colon (Lucas and Blair, 1978; Said et al., 1987). In rat small intestine the ISAM pH ranges from 5.5 (in the duodenum) to approximately 6.5 (in the colon; Lucas and Blair, 1978). This acidic microclimate is maintained by Na+/H+ exchangers on the brush border that pump H+ ions into the lumen creating the acidic microclimate (Thwaites and Anderson, 2007). PCFT works as a folate/H+ symporter, using the gradient created by the Na+/H+ exchanger to bring folate into the cells (Zhao et al., 2009).  7  There are some individuals born with a non-functional variant of PCFT and this genetic disorder is known as hereditary folate malabsorption (HFM; Qiu et al., 2006). Individuals with HFM are unable to absorb a sufficient quantity of folate to meet their daily needs for folate.  These individuals depend on large oral or parenteral doses of FA to meet their folate requirement (Zhao et al., 2009).  When Pcft is knocked out in mice (Pcft -/-), the mutation is not embryonic lethal and the mice develop normally until about 4 weeks of age when severe macrocytic normochromic anemia and pancytopenia develops (Običan et al., 2010). Pcft-/- mice cannot be rescued by oral administration of FA, but an intraperitoneal injection of folate rescues them from the development of megaloblastic macrocytic anemia (Običan et al., 2010).  Pcft-/- mice as well as individuals suffering from HFM provide strong evidence for PCFT as the main route of intestinal folate uptake because both the mice and the humans suffer symptoms of folate deficiency if they are left untreated. Reduced folate carrier (RFC).  Before the identification of PCFT, RFC (SLC19A1) was thought to be the main folate transporter in the small intestine and this is still a point of scientific disagreement (Balamurugan and Said, 2006).  RFC is a member of the SLC family and an anion exchanger.  It transports folate into the cell driven by the energy gradient derived from the downstream transport of organic anions out of the cell (Goldman, 1971). RFC is ubiquitously expressed in human tissues, including the intestine, with higher levels of expression in tissues such as the placenta, liver, leukocytes and central nervous system (CNS) and lower levels of expression in the skeletal muscle and heart (Whetstine et al., 2002). The main function of RFC is cellular uptake of folate, as demonstrated by its ubiquitous expression in human tissue (Whetstine et al., 2002).  8  RFC knockout mice (Rfc-/-) are not viable because of embryonic lethality; the embryos die shortly after implantation (Gelineau-van Waes et al., 2008). However, if the mother receives a daily low-dose of subcutaneous FA injection (25 mg/kg/day), the Rfc-/- embryos may survive past embryonic day 6.5, but they are developmentally delayed along with a number of disorders such as NTDs and failure of hematopoiesis (Gelineau-van Waes et al., 2008). In mothers injected with a daily high-dose subcutaneous FA injection (50 mg/kg/day), 22% of Rfc-/- fetuses survive until embryonic day 18.5. These fetuses are morphologically normal, but have cardiac and lung abnormalities (such as ventricular septal defects, and small, pale lungs with less branching morphogenesis), pale colour and various other defects (Gelineau-van Waes et al., 2008). Because Rfc-/- mice are embryonic lethal it can be inferred that RFC is very important for fetal folate uptake and transport.  Folate receptors (FR).  FR are glycosyl phosphatidylinositol (GPI)-linked proteins that have 3 isoforms, FRα, FRβ and FRγ (FOLR1, FOLR2, and FOLR3, respectively; Antony, 1996). FRα is a membrane-linked form of folate receptor and is differentially expressed in different tissues (Ross et al., 1994). FRα is highly expressed in kidney, lung, ovary and placental tissue, and its expression is greatly elevated in carcinomas of many different tissue types (Ross et al., 1994).  Conversely, FRβ, the most common form of FR, is expressed at lower levels in many normal tissues and carcinomas, but its expression is increased in nonepithelial tumours such as meningiomas and sarcomas (Ross et al., 1994).  FRγ has an alternate mRNA splice site that produces a protein product called FRγ’ (Shen et al., 1994). FRγ has an amino terminal signal peptide for insertion into the 9  membrane but this process may be rendered less efficient due to a carboxyl terminal sequence of hydrophobic amino acids interrupted by two charged amino acids (Shen et al., 1994). FRγ is expressed in tissues containing hematopoietic cells such as the spleen, bone marrow and thymus, and is also found in ovarian, cervical and uterine carcinomas (Shen et al., 1994). A deletion of 2 base pairs from the intact FRγ mRNA results in a truncated version of FRγ, denoted as FRγ’, that lacks an amino terminal signal peptide (Shen et al., 1994) and therefore is not a membrane-anchored protein.  Functionally, FRα facilitates the uptake of folates by receptor-mediated endocytosis (Kamen et al., 1988). Folates bind to the FR forming a FR-folate complex, which is then endocytosed into an acid-resistant endosome (Kamen et al., 1988). Within the endosome, the acidic pH causes the release of folate from the receptor.  Subsequently, folate is thought to be exported into the cytoplasm by PCFT (Anderson et al., 1992; Zhao et al., 2009). The FR is then recycled back to the surface membrane (Kamen et al., 2004; Paulos et al., 2004).  The importance of the FRα versus FRβ is illustrated by their corresponding knockout mice. Folbp1 is the murine equivalent of FRα and Folbp2 is the murine equivalent of FRβ.  Fetuses of Folbp1 knockout mice (Folbp1-/-) develop NTDs whereas fetuses of Folbp2 knockout mice (Folbp2-/-) develop normally with their neural tubes properly closed (Piedrahita et al., 1999). PCFT, RFC and FRα have all been shown to be differentially expressed in human cell lines in the presence of different folate conditions (Ashokkumar et al., 2007; Crott et al., 2008). The relative expression of PCFT mRNA is increased to 125% of the control in Caco2 cells treated with 0.25 µM FA, but is decreased to 50% of the control in cells 10  treated with 100 µM FA (Ashokkumar et al., 2007). Long-term exposure (maintained for 5 passages) of Caco2 cells to 0.25 µM FA increases the relative expression of RFC mRNA to 110% and relative RFC protein levels to 135% compared to the control (Ashokkumar et al., 2007). Treatment of Caco2 cells with 100 µM FA decreases RFC mRNA expression to 80% and RFC protein levels by 50% compared to the control (Ashokkumar et al., 2007). In HK-2 cells, FA treatment at 0.25 µM increases FOLR mRNA levels to125% and FR protein levels to 135% compared to the control (Ashokkumar et al., 2007). Similarly, FOLR mRNA and protein levels are reduced to ~60% in HK-2 cells treated with 100 µM FA compared to the control (Ashokkumar et al., 2007). Clearly, the expression of folate transporters is downregulated at high extracellular concentrations of FA and upregulated at lower concentrations of FA.  PCFT expression appears to be regulated by promoter methylation in human leukemia cell lines (Gonen et al., 2008) as well as by extracellular folate concentrations. A CpG island located in the promoter region of SLC46A1 (PCFT) is 85-100% methylated and correspondingly low levels of PCFT mRNA transcripts and proteins are the result (Gonen et al., 2008). When treated with a demethylating agent (5-Aza-2’-deoxycytidine), expression of PCFT is restored to 50 times of that observed in untreated cells (Gonen et al., 2008). Once folate is absorbed, it is transported across the basolateral membrane entering the portal vein through a transporter-mediated process. The transporters that are thought to play this role include multidrug-resistance-associated protein (MRP) 1-5 (Kruh and Belinsky, 2003) and breast cancer resistance protein (BCRP; Lemos et al., 2008; Maubon et al., 2007). In particular, BCRP is able to export mono-, di- and tri-glutamate forms of 11  folates (Chen et al., 2003). BCRP is expressed in Caco2 cells and also in pooled samples of human small intestine (Maubon et al., 2007). BCRP expression is affected by cellular folate status in Caco2 cells.  For example, in the presence of low extracellular folate conditions (1 nM folate, after 69 days of treatment), BCRP expression is induced 3.9 - 5.7 fold compared to the control (Lemos et al., 2008). BCRP and MRP1-5 are members of the ATP-Binding Cassette (ABC) transporter family. This family of transporters transports folates and other molecules using the chemical energy derived from the hydrolysis of adenosine triphosphate (Stewart et al., 1996; Zhao et al., 2009).   1.1.4 Physiological Functions Folate exerts its physiological role by participating in the transfer of a single carbon unit in cellular reactions (one-carbon metabolism). Thus it plays an important role in DNA synthesis and repair, methylation, amino acid metabolism, and protein metabolism.  Folate participates in DNA synthesis by donating a methyl group from 5,10-methylenetetrahydrofolate to deoxyuridine monophosphate (dUMP) to produce deoxythymidine monophosphate (dTMP; Figure 1.2). Eventually dTMP is incorporated into DNA. During folate deficiency, the ratio of dTMP to dUMP could decrease, resulting in a higher proportion of uracil to thymine in the cell (Blount et al., 1997). This in turn can lead to uracil misincorporation into DNA and possibly DNA damage, such as double strand breaks (Blount et al., 1997).  A second important role for folate is in DNA methylation: 5MTHF donates a methyl group to cobalamin (vitamin B12, a cofactor for methionine synthase) to form methyl-cobalamin, which subsequently donates the methyl group to homocysteine producing 12  methionine (Figure 1.2). Methionine is then converted to S-adenosylmethionine (SAM), which can donate the methyl group directly to DNA. DNA methylation occurs predominantly on a cytosine residue where it is followed by a guanine residue (CpG; Cedar, 1988). DNA methylation in the promoter regions of genes plays a role in regulating gene expression (Rountree et al., 2001; Siegfried and Simon, 2010).  As well, folate participates in purine synthesis via transferring a formyl group from 10-formylfolate to phosphoribosyl glycinamide to produce inosine monophosphate, which is then used in purine synthesis (Figure 1.2). Purines (nucleotides such as deoxyguanosine monophosphate and deoxyadenosine monophosphate) are important for the synthesis of DNA and RNA.  In addition, folate is also involved in the metabolism of some amino acids, such as the conversion of serine to glycine (Figure 1.2) and the synthesis of methionine from homocysteine as described above.  1.1.5 Interactions with Vitamin B12  A potentially negative nutrient-nutrient interaction between folate and vitamin B12 is that a deficiency of vitamin B12 can induce a functional folate deficiency (Vilter et al., 1963).  The biochemical basis of this interaction is that 5,10-methylene THF (Tetrahydrofolate) is converted to 5MTHF, a one-way reaction catalyzed by 5,10-methyleneTHF reductase (MTHFR; Figure 1.2).  5MTHF is then converted to THF, a reaction catalyzed by methionine synthase with vitamin B12 acting as a co-enzyme (Figure 1.2). In the absence of vitamin B12, folate is trapped as 5MTHF with a diminished cellular pool of THF, a phenomenon known as the ‘methyl trapping hypothesis’ (Sauer 13  and Wilmanns, 1977).  Consequently, less THF is available to support normal DNA synthesis and impaired methylation can occur due to a diminished synthesis of SAM, a major cellular methyl donor. Clearly, if megaloblastic macrocytic anemia is developed due to a deficiency in folate, vitamin B12, or a combination of both, treatment with just folate can alleviate the anemia by normalizing DNA synthesis; however, if the anemia is caused by vitamin B12 deficiency, methylation remains impaired leading to the development of irreversible neurological consequences. Data from the NHANES (1999-2002) survey for seniors (≥60 years) revealed that seniors with low serum vitamin B12 (Serum vitamin B12 <148 pmol/L or serum methylmalonic acid >210 nmol/L) and high serum folate (>59 nmol/L; the 80th percentile) were more likely to have anemia and cognitive impairment (Morris et al., 2007; Selhub et al., 2009). Thus care must be taken not to mask a potential vitamin B12 deficiency with high doses of folate when treating megaloblastic macrocytic anemia that could lead to cognitive decline.    1.1.6 Deficiency Folate deficiency increases the risk of hyperhomocysteinemia and megaloblastic macrocytic anemia (in severe cases). Folate deficiency has also been linked to the development and progression of certain types of cancer. The incidence of NTDs such as spina bifida and anencephaly can be lowered by ensuring women of a child bearing age take FA supplements pre- and peri-conceptionally (MRC Vitamin Study, 1991; Berry et al., 1999). Studies have also found that the risk of other birth defects (such as cleft palate or cleft lip) appear to decrease when mothers increase their folate intake (Czeizel et al., 1999; van Rooij et al., 2004). Prevention of 14  folate-related NTDs was the main reason for the decision to fortify grains and flours with FA in Canada. Health Canada’s stance is that it is important that women of childbearing age have an adequate folate status to prevent NTDs, especially in the case of unplanned pregnancies where prenatal vitamins may not be used. Therefore women of childbearing age are directed by Health Canada to take a supplement containing 400 μg FA daily, over and above the added FA in food.  Hyperhomocysteinemia is a condition that has been linked to an increased risk for ischemic heart disease and stroke (The Homocysteine Studies Collaboration, 2002). At the metabolic level, homocysteine plays a role in the development of endothelial dysfunction and atherothrombosis by inducing proinflammatory factors, oxidative stress and endoplasmic reticulum stress (Austin et al., 2004). Heatlhy people aged 18-59 years with a normal fasting plasma homocysteine level (9.5 ± 0.9 µmol/L) were given oral L-methionine (10, 25, 100 mg/kg) or lean chicken (551 ± 30 g, containing 3.2 ± 0.2 g methionine) and their plasma homocysteine response and brachial artery flow dilatation was measured (Chambers et al., 1999). After 4 hours, dose-dependent increases of plasma homocysteine and decreases in brachial artery flow-mediated dilatation were observed, indicating endothelial dysfunction (Chambers et al., 1999). In one-carbon metabolism, homocysteine accepts a methyl group from 5MTHF via vitamin B12 to produce methionine, which is subsequently converted to SAM (Figure 1.2). Thus folate deficiency can result in hyperhomocysteinemia. Folate deficiency-induced hyperhomocysteinemia can be reversed by administration of FA (Ubbink et al., 1994). It has also been shown that folate supplementation can lower plasma homocysteine. The Heart Outcomes Prevention Evaluation (HOPE) 2 is a randomized, 15  double-blind, placebo-controlled trial investigating the effect of a B vitamin mix (2.5 mg FA, 50 µg vitamin B6, and 1 mg vitamin B12) on the reduction of cardiovascular events in patients (≥ 55 years) with prior vascular disease or diabetes (HOPE-2 investigators, 2006). After an average of 5 years of the study, mean plasma homocysteine decreased in the treatment group and increased in the placebo group, while treatment did not reduce the primary outcomes (death from cardiovascular causes, myocardial infarction, and stroke) in the treatment group compared to the placebo group (HOPE-2 investigators, 2006).  The Homocysteine Lowering Trialists’ Collaboration performed meta-analyses of randomized controlled trials assessing the effects of FA supplements on blood homocysteine concentrations (1998) and to determine the lowest amount of FA associated with the highest reduction in plasma homocysteine concentrations (2005). The first meta-analysis determined that folic acid supplementation at any doseage (0.5 – 5 mg/d) resulted in an approximate 25% reduction in plasma homocysteine (Homocysteine Lowering Trialists’ Collaboration, 1998). The second meta-analysis determined that supplementation with ≥ 0.8 mg FA daily produced the maximum reduction in plasma homocysteine (23%, 95% CI: 21 – 26%; Homocysteine Lowering Trialists’ Collaboration, 2005). As well as lowering plasma homocysteine, folate supplementation has been shown to improve endothelial dysfunction. 29 healthy subjects (aged 40 – 70 years) with hyperhomocysteinemia (mean: 9.0 ± 1.7 µmol/L, at baseline) received FA (10 mg/d) for 1 year (Woo et al., 2002). FA supplementation resulted in higher plasma folate levels (40 ±5 vs. 24 ± 5 nmol/L, p<0.001), lower total plasma homocysteine levels (9.0 ± 1.7 vs 7.9 ± 2.0 µmol/L, p<0.001) and improved flow-mediated endothelium-dependent 16  dilatation of the brachial artery (8.9 ± 1.5% vs 7.4 ± 2.0%, mean difference=1.5%; 95% CI: 1-2%; p<0.0001), compared to the baseline (Woo et al., 2002). Folate deficiency can also lead to the development of megaloblastic macrocytic anemia (Herbert, 1964) due to altered DNA synthesis and impaired repair mechanisms (Das and Hoffbrand, 1970). Decreased synthesis of thymidine during folate deficiency can lead to the production of red blood cells that are not fully matured (Koury et al., 1997). As a result of impaired thymidylate synthesis and DNA synthesis, the blood cells continue to grow without dividing, resulting in large, immature red blood cells (Koury et al., 1997; Vilter et al., 1963). Megaloblastic macrocytic anemia can be caused by a deficiency in folate alone or a combined deficiency of vitamin B12 and folate (Vilter et al., 1963), as discussed in the previous section.  1.1.7 Neural Tube Defects and Folic Acid Fortification   NTDs affect 0.5-2 births/1,000 live births (Greene et al., 2009). They are caused by a failure of the neural tube to close (primary neurulation) during embryogenesis (Blom, 2009). In humans, primary neurulation occurs 3-4 weeks post conception (Blom, 2009). The process of neurulation occurs before most women even become aware of their pregnancy. A study conducted by the MRC Vitamin Study Research Group found that administration of 4 mg of FA daily to women before and up to 12 weeks of pregnancy results in a 72% reduction in the incidence of NTDs compared to the women given a placebo (MRC Vitamin Study Research Group, 1991). Another study by Berry et al. (1999) reported a 79% decrease in the rate of NTD-affected pregnancies in women from 17  a northern region in China who took a 400 μg FA supplement daily starting after last menstrual period compared to women who did not take a daily FA supplement.  Mandatory FA fortification was introduced in Canada in 1998 at the level of 150 μg FA / 100 g flour or 200 μg FA / 100 g pasta (Public Health Agency of Canada, 2004). The fortification levels are designed so that the average person receives approximately 100 μg FA daily from the consumption of fortified foods (Public Health Agency of Canada, 2004).  This mandatory FA fortification is implemented to ensure that the majority of women of childbearing age will have adequate folate status due to strong evidence that pre- and peri-conceptional FA supplementation reduces the incidence of NTDs (MRC, 1991; Berry et al., 1999). Unfortunately, the mechanism by which FA reduces the incidence of NTDs remains to be elucidated.  In addition, FA fortification has also been implicated in lowering colon cancer incidence (Giovannucci et al., 1998; Su and Arab, 2001; La Vecchia et al., 2002) and the potentially lowering the risk of cardiovascular disease by decreasing plasma homocytsteine levels (Homocysteine Lowering Trialists’ Collaboration, 1998 and 2005; Brouwer et al., 1999). There are some concerns associated with FA fortification. Because of the structure of FA and its metabolism, FA enters the folate cycle as THF (Figure 1.1 & 1.2).  As discussed earlier, THF is metabolized to 5,10-methyleneTHF, which is required for the synthesis of dTMP and subsequently DNA synthesis. Thus, FA fortification provides a means to bypass an integral step in the vitamin B12-dependent folate recycling. Consequently, FA prevents vitamin B12 deficiency-induced megaloblastic anemia while disguising vitamin B12 deficiency-induced neurological lesions (Vilter et al., 1963). 18  The rate of NTD-affected pregnancies in Canada had dropped from 1.58 / 1,000 births before FA fortification to 0.86/1,000 births after the implementation of FA fortification in 1998 (De Wals et al., 2007). Since FA fortification is designed for women of child-bearing age, the health impacts of long-term high FA intakes on other subpopulations in our society is largely unknown.  1.2 Wnt Signalling Pathway 1.2.1 Overview  Wingless-type proteins (Wnts) are a family of secreted glycoproteins that are involved in the Wnt signalling pathway (Moon et al., 2004). A number of recombinant and native Wnt proteins are glycosylated before secretion in several murine cell lines (Smolich et al., 1993). This pathway is responsible for many developmental processes (e.g. closure of the neural tube during neurulation and convergent extension during gastrulation in zebrafish (Ungar et al., 1995) and Xenopus (Moon et al., 1993), homeostasis of bone mass (Baron et al., 2006), and neurogenesis in adults (Malaterre et al., 2007). In addition, dysregulation of Wnt signalling has been implicated in the development of a wide range of diseases, for example, hereditary and sporadic colon cancer  (Bienz and Clevers, 2000), birth defects (Zeng et al., 1997; Hamblet et al., 2002; Carter et al., 2005) and nervous system disease (Freese et al., 2010). Wnt was first identified as int1, an activated proto-oncogene involved in the aberrant formation of tumors in mice infected with the mouse mammary tumour virus (Nusse and Varmus, 1982). This was also the beginning of research into the involvement of Wnt in cancer development (Polakis, 2011). 19   From its humble beginnings as wingless and int1, Wnt is now known to play the part of a secreted signalling molecule in the Wnt signalling pathway. ‘Wnt signalling pathway’ is a general term covering two main distinct pathways: the canonical Wnt signalling pathway or β-Catenin-dependent Wnt signalling pathway and the non-canonical Wnt signalling pathway or β-Catenin-independent Wnt signalling pathway.  The β-Catenin-independent Wnt signalling pathways includes the planar cell polarity (PCP) pathway and the Wnt/Ca2+ pathway (Chien et al., 2009).     1.2.2 β-Catenin-dependent Wnt Signalling Pathway The canonical Wnt/β-Catenin-dependent signalling cascade is initiated when a secreted Wnt protein binds to the frizzled (FZD) receptor and low density lipoprotein receptor-related protein 5/6 (LRP5/6), the co-receptor (Figure 1.3). The binding of both FZD and LRP5/6 by Wnt is necessary for signal transduction (Tamai et al., 2000).  FZD acts as a G-protein coupled receptor when it is activated upon Wnt binding (Katanaev, 2010). Katanaev (2010) has shown that the trimeric G protein Go binds to FZD in the presence of Wnt. The βγ subunit of Go activates dishevelled (DSH).  DSH and the αo subunit then converge to inhibit axin. The inhibition of axin prevents the phosphorylation and eventual destruction of β-Catenin (Katanaev, 2010), allowing β-Catenin to move into the nucleus.  In the nucleus, β-Catenin complexes with transcription factors of the T cell factor (TCF) and lymphoid enhancer-binding factor (LEF) families, exerting its effect on the transcription of target genes. When this pathway is not stimulated by Wnt, antagonized, or blocked, axin is not inhibited by DSH. Axin, along with glycogen synthase kinase 3 (GSK3) and adenomatous polyposis coli (APC), then 20  bind to and phosphorylate β-Catenin. This phosphorylation marks β-Catenin for ubiquitination and subsequent degradation by the proteosome.  Wnt proteins are post-translationally modified in the endoplasmic reticulum (ER) before extracellular release (Komiya and Habas, 2008). Wnt proteins, specifically murine Wnt3a, have also been shown to be palmitoylated (Willert et al., 2003) and palmitoleated (Takada et al., 2006). Mutation of the palmitoylation site in Wnt proteins leads to loss of signalling activity (Willert et al., 2003; Galli et al., 2007; Komekado et al., 2007; Kurayoshi et al., 2007).  In contrast, mutation of the palmitoleation site inhibits secretion and results in a buildup of the mutated Wnt protein in the ER (Takada et al., 2006).  Thus, these two lipid modifications in Wnt proteins are responsible for the hydrophobicity of Wnts and are required for both signalling and secretion (Harterink and Korswagen, 2012). Post-translational modifications of Wnt proteins appear to be mediated by Porcupine (Porc), a member of the membrane-bound O-acyltransferase family (Harterink and Korswagen, 2012). In Drosophila, Porc is localized to the ER at the same location that Wnt modification is thought to take place (Zhai et al., 2004). In Drosphila, Porc interacts with the N-terminal domain of wg (in a region that is also conserved in Wnt proteins) and is essential to the process of N-glycosylation because it anchors wg/Wnt to the ER membrane for subsequent glycosylation (Tanaka et al., 2002). If Porc is depleted, Wnt secretion is inhibited and a buildup of Wnt proteins in the ER occurs (van den Heuvel et al., 1993).  Wnt sorting receptors (Wntless; Wls) are also necessary for Wnt secretion (Harterink and Korswagen, 2012). Wls proteins are highly conserved 7-pass 21  transmembrane proteins (Banzinger et al., 2006). When Wls binds to Wnt as illustrated by co-immunopreciptation experiments and in cells with non-functioning Wls, Wnt proteins build up in the cell (Banzinger et al., 2006; Bartscherer et al., 2006). Further analysis of non-functioning Wls proteins in Drosophila reveals that Wnt builds up in the Golgi (Harterink and Korswagen, 2012). Thus Wls probably functions in Wnt secretion from cells by acting as a shuttle, transporting Wnt proteins from the Golgi apparatus to the plasma membrane of cells (Harterink and Korswagen, 2012). In a model proposed by Harterink and Korswagen (2012), Wnt proteins are post-translationally modified, which requires Porc, and form a complex with Wls in the ER.  The Wnt-Wls complex is then translocated to the Golgi apparatus and secreted from the Golgi in secretory vesicles. Wnt is translocated to the plasma membrane and then secreted into the extracellular space via exocytosis. Wnt proteins appear to be released from Wls by endosomal acidification en route to the plasma membrane, as inhibition of V-ATPase prevents β-Catenin-dependent and independent signalling from occurring (Coombs et al., 2010). Inhibitors of acidification increase intracellular Wnt, Wls and Wnt-Wls complex levels and inhibit the release of Wnt from the Wnt-Wls complex (Coombs et al., 2010). β-Catenin-dependent Wnt signalling is constitutively inhibited by the β-Catenin destruction complex, made up of GSK3, casein kinase I (CKI), axin, and APC (Ikeda et al., 1998; Archbold et al., 2012). This complex binds to β-Catenin resulting in GSK3-dependent phosphorylation of β-Catenin, marking it for ubiquitination and subsequent destruction by the proteasome (Ikeda et al., 1998; Archbold et al., 2012). This pathway can also be inhibited by secreted proteins that interfere with or compete for receptor binding (Komiya and Habas, 2008). These secreted proteins include Dickkopf (DKK; 22  Glinka et al., 1998), Wnt-inhibitor protein-1 (WIF-1; Hsieh et al., 1999), soluble frizzled-related proteins (SFRPs; Hoang et al., 1998) and FrzB (Frizzled motif associated with bone development; Lin et al., 1997; Wang et al., 1997). DKK proteins bind to LRP5/6, the co-receptor for FZD, preventing the association of the two proteins and subsequently β-Catenin-dependent Wnt signalling (Bafico et al., 2001). WIF-1, SFRP and FrzB are examples of Wnt signalling inhibitors that bind to Wnt proteins and inhibit Wnt signalling regardless of dependence on β-Catenin (Kawano and Kypta, 2003). SFRPs (including FrzB, otherwise known as SFRP3) contain an N-terminal cysteine-rich domain (CRD) that shares 30-50% similarity to the CRD found in FZD proteins (Melkonyan et al., 1997). WIF-1 has a WIF domain and 5 epidermal growth factor repeats (Hsieh et al., 1999). This WIF domain is similar to an extracellular domain of RYK, a tyrosine kinase that was later discovered to have a role in Wnt signalling in Drosophila (Patthy, 2000).   1.2.3 β-Catenin-independent Wnt Signalling Pathways The planar cell polarity pathway (PCP pathway) is a β-Catenin-independent Wnt signalling pathway first observed in Drosphila that appears to be conserved in mammalian tissue (Mlodzik, 2002; Veeman et al., 2003). As the classification implies, PCP signalling is mediated through the binding of Wnt to a receptor and transduction through DSH, but β-Catenin is not involved. Signalling in the PCP pathway occurs when a Wnt (Wnt4, Wnt5a or Wnt11; Komiya and Habas, 2008) protein binds to FZD. This binding does not involve LRP5/6 as in the canonical pathway. As a result of the binding, DSH is activated and complexes with Daam1 (Dishevelled associated activator of morphogenesis), leading to the activation of Rho (small G protein) and ROCK (Rho 23  associated kinase). The PCP pathway is especially important in tissues that have a defined polarity, for example, stereocilia in the inner ear, organization of hair follicles (Wang et al., 2007) and convergent extension during gastrulation (Keller et al., 2003).  The Wnt/Ca2+ pathway is another β-Catenin-independent signalling pathway that shares components with the PCP pathway, but is distinct enough that it is categorized as the third Wnt signalling branch (Komiya and Habas, 2008). The role of Ca2+ as a second messenger in the Wnt pathway is realized when the frequency of calcium transients in the enveloping layer of the blastodisc of zebrafish embryos is doubled in response to injections of Wnt5a (Slusarski et al., 1997) and Wnt11 (Westfall et al., 2003) mRNA. In this pathway, Wnt binding to FZD and possibly a co-receptor (Ror2 or Kynpek) results in the activation of calcium/calmodulin-dependent kinase II (CamKII; Kühl et al., 2000) and protein kinase C (PKC; Sheldahl et al., 1999). Binding of Wnt to FZD receptors can also activate phospholipase C (PLC) and phosphodiesterase (PDE) via G protein binding (Kohn and Moon, 2005).  The Wnt/Ca2+ pathway is important for body plan specification (Kühl et al., 2000) and it seems to have a role in slow twitch muscle fibre formation in adults (Naya et al., 2000). An increase in intracellular calcium release and PLC signalling results in the downstream activation of cdc42, a regulator of cell adhesion, migration and tissue separation (Choi and Han, 2002). Intracellular calcium increase activates CamKII, which activates TAK1 and NLK.  Activation of TAK1 and NLK in turn causes phosphorylation of TCF and LEF, interfering with binding of these transcription factors to DNA (Ishitani et al., 1999). In this way Wnt/Ca2+ signalling can inhibit the canonical Wnt signalling 24  pathway and Wnt5a, a stimulator of the non-canonical pathway, can act as a tumour suppressor (Ishitani et al., 1999).  1.3 Linking Folic Acid and Wnt Signalling Pathways to Colorectal Cancer 1.3.1 Folic Acid and Colorectal Cancer FA fortification has been implicated in both the development of and the protection against colon cancer. There are animal studies as well as epidemiological studies that show a protective role of folate against colorectal cancer. However, there have also been animal and epidemiological studies showing the opposite, that higher folate intake promotes cancer development. The mechanism behind folate’s potential role in cancer protection or promotion is not yet elucidated, however the timing of folate exposure appears to play an important role in whether folate will be protect against or promote cancer (Ulrich and Potter, 2007).   1.3.1.1 Protective Effects of Folate Intakes Against Cancer There are both animal studies and epidemiological studies showing the protective effect of folate against cancer. A study with Sprague-Dawley rats fed diets containing either 0 or 8 mg/kg FA found that folate deficiency promotes the development of colonic neoplasia (2/7 rats vs. 7/7 rats) and carcinoma development (1/7 rats vs. 6/7 rats) after 20 weeks of treatment with dimethylhydrazine, a colon carcinogenic (Cravo et al., 1992).   Another study had similar results using the same animal model of colorectal cancer: microscopic colorectal neoplastic foci were induced in Sprague-Dawley rats by treatment with dimethylhydrazine (Kim et al., 1996). This study used the same diets as 25  the Cravo et al., (1992) study, and found that increasing levels of dietary folate up to 8 mg/kg diet, decreases the percentage of rats with macroscopic tumours (p<0.03) and the average number of tumours per rat (p<0.04; Kim et al., 1996). In rats fed a high folate diet (40 mg folate/kg diet), there was a nonsignificant trend towards increasing macroscopic tumour carcinogenesis compared to the other groups (Kim et al., 1996). In humans, pooled analyses of 13 prospective cohort studies from Canada, the US, Netherlands and Sweden investigating folate intake and the incidence of colon cancer found that folate intake in the highest quintile is mildly protective against colon cancer when compared to the lowest quintile of intake (0.92 risk ratio (RR); 95% CI 0.84-1.00 for dietary folate and 0.85 RR; 95% CI 0.77-0.95 for dietary and supplemental folate; Kim et al., 2010). Similarly a meta-analysis of cohort studies and case-control studies from Canada, the US, Netherlands, Italy, France, Finland and Switzerland examining the relationship between folate intake and colorectal cancer risk also found that folate at the highest intake (The highest tertile, quartile or quintile from the studies in the meta-analysis: ranging from >249 µg/day to >2430 µg/day) is protective compared to the lowest intake (The lowest tertile, quartile, or quintile: ranging from <103 µg/day to <301 µg/day; 0.75 RR; 95% CI 0.64-0.89 for intake from foods alone and 0.95 RR; 95% CI .81-1.11 for intake from foods and supplements; Sanjoaquin et al., 2005). The meta-analysis shows that there is more protection from naturally occurring folate in food as opposed to FA from fortified foods and supplements.  The Nurses’ Health Study (1980-1994) reported that women who have taken FA-containing vitamins for less than 15 years have no reduction in the risk of colon cancer across the ranges of folate intake (≤200, 201-300, 301-400, and >400 µg/d), but women 26  have a 31% reduced risk (RR of 0.69, 95% CI 0.52-0.93) if they consume >400 µg folate/d compared to intakes of  ≤200 µg folate/d (Giovannucci et al., 1998). Another study examining the latency between folate intake and colorectal cancer risk found that high folate intakes (≥ 800 µg/d) for 12-16 years before cancer diagnosis offers protection compared to low folate intakes (<250 µg/d; RR 0.69; 95% CI 0.51-0.94); however, folate intake during the 4 years prior to the study is not associated with colorectal cancer risk (Lee et al., 2011). The Nurses’ Health Study and the Health Professionals Follow-up Study has also found that both long-term (8 – 16 year prior to diagnosis) and short-term (0 – 8 years prior to diagnosis) total folate intake before diagnosis offers protection against colorectal adenomas comparing high (≥800 µg/d) and low folate intakes (<250 µg/d; Lee et al., 2011).  The strongest inverse association was observed with high total folate intake (≥800 µg/d) compared to low intake (<250 µg/d) 4-8 years before adenoma diagnosis (Odds Ratio (OR) 0.68; 95% CI 0.60 – 0.78; Lee et al., 2011). Lee’s study (2011) shows that colorectal cancer and colorectal adenomas do not have the same risk factors, as it seems that shorter term folate exposure is important for adenoma risk and longer term folate exposure is important for colorectal cancer risk. These studies build a solid case for the protective effect of adequate folate status. However it cannot be concluded from these studies that folate deficiency contributes to colorectal carcinogenesis.  1.3.1.2 Promoting Effects of Folate Intake on Cancer Development While it has been shown in Sprague-Dawley rats that folate deficiency promotes the development of colorectal neoplasia, another study shows that dietary folate 27  deficiency is protective. For example, folate deficiency protects Sprague-Dawley rats against azoxymethane (AOM)-induced intestinal tumors (Le Leu et al., 2000). In this study weanling rats were fed diets containing either 0 or 8 mg FA/kg for 4 weeks, at which point AOM was administered (Le Leu et al., 2000). After 26 weeks, rats fed the 0 FA diet have significantly lower incidence of total intestinal tumours compared to rats fed diets with the control diet containing FA (p < 0.01; Le Leu et al., 2000). Rats fed the 0 FA diet also have fewer adenocarcinomas than the control (p < 0.01) and show a 71% decrease in tumour malignancy (Le Leu et al., 2000). According to Kim (2004), this study did not actually show that 0 FA diet was protective against colorectal cancer. The animals were fed a casein-based diet, containing 20% fat and measurable levels of folate (Kim, 2004). The colonic folate concentrations were much higher than usual levels achieved by the folate levels used in amino acid defined diets, suggesting the rats in the study were actually exposed to supraphysiologic doses of FA, and this is further supported by the low plasma homocysteine values of the rats in the study (Kim, 2004).  Another study in Sprague-Dawley rats examined the link between maternal dietary folate supplementation and post-weaning dietary folate supplementing in rat pups with AOM-induced colorectal cancer (Sie et al., 2011). This study found that pups born from mothers fed with the control diet (2 mg FA/kg diet) whose diets were supplemented with FA (5 mg/kg diet), had higher tumour multiplicity and burden than rat pups in other diet groups (Sie et al., 2011). The authors theorized that pups born from mothers fed the control diet possibly developed more microscopic precursor lesions in the colorectum (Sie et al., 2011).  28  A study focusing on the link between development of aberrant crypt foci (ACF; an early precursor to colorectal cancer) and FA supplementation revealed that male Sprague-Dawley rats with AOM-induced ACF, develop 54% more ACF (p=0.011) when their diet is supplemented with 8 mg FA/kg diet compared to the 0 folate control-diet fed animals (Lindzon et al., 2009). This study also revealed a correlation between folate supplementation and tumour multiplicity (r=0.32, p=0.002), tumour burden (r=0.35, p=0.001), and rectal epithelial proliferation (r=0.39, p<0.001) in Sprague-Dawley rats with AOM-induced colorectal cancer (Lindzon et al., 2009). From their study, Lindzon et al. (2009) concluded that FA supplementation after treatment with AOM, may promote the progression of ACF to tumours. A recent study looking at unmetabolized FA in the serum of participants in NHANES studies from 1999-2002 (2 cycles) and serum folate concentrationss in participants from 5 cycles of NHANES studies (1999-2008) found that in adults older than 60, those whose serum did not contain unmetabolized FA had a 24% reduced risk of developing all cancer (Bauldauff, 2013). The study also found that individuals in the highest quartile of serum folate (quartiles not defined) were 1.4 times more likely to have cancer than those with lower serum folate concentrations (Bauldauff, 2013). Women over age 60 in the highest quartile of serum folate were 1.9 times more likely to have breast cancer than women in lower quartiles (Bauldauff, 2013). Another study found that among healthy, post-menopausal women, those with detectable plasma FA had natural killer cell cytoxicity 23% lower than those without detectable plasma FA (Troen et al., 2006). Furthermore, among women who consumed a folate-rich (> 233 µg folate/d) diet and consumed supplements containing > 400 µg FA/d, natural killer cell cytoxicity was lower 29  compared with women who consumed a low-folate (<233 µg folate/d) diet and used no FA supplements (Troen et al., 2006). These two studies in older adults show that more research needs to be done on the effect of FA on the body and on cancer.   A study looking at American (The US Surveillance, Epidemiology and End Result registry, 1986 - 2002) and Canadian (Canadian Cancer Statistics, 2006) colon cancer incidence show a decline in the incidence of colorectal cancer in both the US and Canada; however this downward trend was interrupted by a sudden upturn starting in 1996 for the US and 1997 for Canada (Figure 1.4; Mason et al., 2007). Hirsch et al. (2009) observed similar trends to the US and Canada when examining pre- and post-FA fortification colorectal cancer incidence in Chile. Data, including length of hospital stay, diagnosis and discharge, showed that in patients over age 45, colon cancer-caused hospital discharges are increased (45-64 years: rate ratio 2.61; 99% CI 2.58-2.93; 65-79 years: rate ratio 2.9; 99% CI 2.86-3.25) during the post-fortification period (2002-2004) compared to pre-fortification (1992, 1993 and 1996; Hirsch et al., 2009). In a Norwegian intervention study, the hazard ratio (HR) for developing any cancer is 1.21 (95% CI 1.03-1.41) for participants who received a multivitamin supplement, containing FA (800 µg/day), vitamin B12 (400 µg/day) and vitamin B6 (40 mg/day), or FA (800 µg/day) and vitamin B12 (400 µg/day) compared to patients who received vitamin B6 (40 mg/day) alone or a placebo, suggesting that FA increases the overall risk of getting any cancer (Ebbing et al., 2009). Furthermore, participants receiving FA and vitamin B12 also have increased risk of cancer death (HR, 1.38; 95% CI 1.07-1.79) and all-cause mortality (HR, 1.18; 95% CI 1.04-1.33; Ebbing et al., 2009). It appears that FA may play a role in both preventing and promoting cancer development depending on the timing of exposure.  30  1.3.1.3 Possible Mechanisms of Folate’s Role in Colorectal Cancer The proposed mechanisms of colorectal carcinogenesis due to insufficient folate intake or status include decreased production of SAM for DNA methylation causing global DNA hypomethylation (Kim, 1999) and the potential for misincorporation of uracil during DNA synthesis (Blount et al., 1997). Cell culture studies with human colonic epithelial cells have shown that folate deficiency causes increased uracil incorporation into DNA, increased DNA double strand breaks, impaired cellular response to oxidative and alkylation damage and decreased global DNA methylation (Duthie et al., 2000). Folate deficiency altered the expression of genes involved in cell cycle control (e.g. p53), DNA repair, apoptosis and angiogenesis (e.g. VEGF) in a cell-specific manner in HCT116, Caco2, HT29 and LS513 colon cancer cells (Novakovic et al., 2006).  In Caco2 cells, p53, which is involved in the regulation of apoptosis and cell cycle, and VEGF, which is involved in angiogenesis, are both upregulated after 20 days culture in folate deficient conditions (0 folate RPMI 1640 plus 10% dialyzed fetal bovine serum containing approximately 0.6 nM folate) relative to higher folate conditions (2.3 µM FA RPMI 1640 plus 10% fetal bovine serum); however, expression of these genes is down regulated in HCT116 cells cultured in folate deficient conditions (Novakovic et al., 2006). In people at high risk of developing colon cancer due to their history of developing recurrent adenomatous colorectal polyps, FA supplementation at 2 mg/day reduces proliferation of cells obtained from rectal biopsies compared to that in the placebo control group (Khosraviani et al., 2002). Cell culture studies are generally consistent with prospective cohort studies showing that folate deficiency is associated 31  with higher risk for colorectal cancer development whereas supplementation seems to protect against it. Many epidemiologic studies point to a protective role for folate; however studies have linked rises in colon cancer incidence to the initiation of a FA fortification program. It is theorized that the timing of folate exposure can predict whether folate is protective or it promotes colorectal cancer (Ulrich and Potter, 2007). Timing refers to whether the patient has precancerous lesions, polyps, adenomas, or neoplastic foci during the exposure to folate (Ulrich and Potter, 2007). In ApcMin mice, a model of mice genetically predisposed to developing small intestintal and colonic adenomas, folate deficiency (0 mg FA/kg diet) reduced the occurrence of ileal polyps by 62-76% at 6 months compared to the other folate supplemented diets (2, 8, and 20 mg FA/kg diet; Song et al., 2000). In this model of mouse, 3 months is the time of maximum tumour development and 6 months is considered the point at which the tumours are established (Song et al., 2000). The number of ileal polyps was also correlated to serum folate levels in the mice after 6 months (r=0.44, p=0.006), suggesting that after tumours are established in the mice, folate deficiency causes a regression of the ileal polyps in ApcMin  mice (Song et al., 2000). Considering the role of folate in DNA synthesis, it makes sense that restricting folate in rapidly proliferating tissue would cause growth to slow down or stop. A previously discussed study in Sprague-Dawley rats investigating maternal and post-weaning folate supplementation showed that pups from mothers fed the control diet (2 mg FA/kg diet) that were given the FA supplemented diet (5 mg/kg diet) develop higher tumour multiplicity and burden than any other group (Sie et al., 2011). The 32  explanation given for this observation was that pups from mothers fed the control diet may have had more microscopic precursor lesions in the colorectum than pups from dams fed the supplemented diet (Sie et al., 2011). The pups fed the supplemented diet then developed more tumours because the FA promoted tumourigenesis (Sie et al., 2011). This is an example where timing of folate exposure can cause FA to promote tumourigenesis. In fact, this study also shows that maternal supplementation significantly reduces the risk of colorectal adenocarcinomas in rat pups with AOM-induced colorectal cancer (OR 0.36; 95% CI 0.18-0.71, p=0.003; Sie et al., 2011). This study showed that timing of folate exposure is very important, as folate supplementation in utero ias protective against AOM-induced colorectal cancer, whereas post-weaning supplementation does not offer protection, and in certain cases, increases tumour multiplicity and burden (Sie et al., 2011). A randomized, double-blind, placebo-controlled study conducted on patients with a recent history of colorectal adenomas, but no previous invasive large instestine carcinoma, found that FA supplementation (1 mg/d) had higher risks for having 3 or more adenomas and development of noncolorectal cancers compared to the placebo group (Cole et al., 2007). This study had two follow-up intervals, at the second follow-up interval, participants receiving folic acid were more likely to develop multiple adenomas (≥3; RR: 2.32, 95% CI: 1.23-4.35, p<0.02) and more likely to have advanced lesions (RR: 1.67, 95% CI: 1.00-2.80, p<0.05) compared to the placebo group (Cole et al., 2007). The patients in this study had a history of adenomas, so FA supplements were intended to treat secondary occurrences. In this case, FA appeared to promote carcinogenesis, rather 33  than protect against it in patients at higher risk for colorectal cancer. This makes sense as rapidly proliferating tissue requires folate for nucleotide synthesis. The two aforementioned studies in ApcMin mice (Song et al., 2000) and in people with a recent history of colorectal adenomas (Cole et al., 2007), show how timing affects the outcome of folate on carcinogenesis. Since a significant proportion of the population over age 50 have colorectal polyps, the issue of folate exposure and timing is very important. One study conducted at 3 American medical centres found that 50.4% of the asymptomatic adults (aged 50-79 years) in the study had polyps (Pickhardt et al., 2004). In subjects with otherwise normal healthy tissues, folate deficiency appears to predispose them to cancer (Kim, 2004). Folate is important for DNA synthesis, stability, repair and integrity, so it stands to reason that deficiency could compromise DNA allowing DNA errors to go uncorrected and cancer to occur (Kim, 2004). So timing of exposure to folate seems to be an important key in predicting how folate will affect carcinogenesis.  1.3.2 Wnt Signalling and Colorectal Cancer Wnt was initially identified as a protooncogene (Nusse and Varmus, 1982). Later it was discovered that APC, a tumour suppressor located on chromosome 5, is mutated in familial adenomatous polyposis (FAP), an inherited colorectal cancer (Bodmer et al., 1987; Leppert et al., 1987; Solomon et al., 1987). In many cases of sporadic colon cancer both APC alleles are inactivated (Bienz and Clevers, 2000). APC as part of the β-Catenin destruction complex binds with β-Catenin (Su et al., 1993) and the formation of the complex leads to the destabilization of β-Catenin.  Therefore, APC is posited as a 34  negative regulator of β-Catenin-dependent Wnt signalling (Munemitsu et al., 1995). In colorectal cancer where APC is inactivated, β-Catenin is stabilized resulting in constitutive activation of Wnt signalling (Morin et al., 1997; Korinek et al., 1997). Axin is another member of the β-Catenin destruction complex (Ikeda et al., 1998) that is also mutated in some colorectal cancer cases (Fearnhead et al., 2004; Suraweera et al., 2006). Patients with mutation to AXIN2 are predisposed to developing colon cancer (Lammi et al., 2004). Loss of function alterations to components of the β-Catenin destruction complex leads to accumulation of stabilized β-Catenin within the cytoplasm.  Subsequently, -Catenin enters the nucleus where it forms a complex with the transcription factors TCF/LEF and binds to DNA, activating transcription of target genes (Bienz and Clevers, 2000). β-Catenin mutations can also occur in colorectal cancer (Fearnhead et al., 2004), most commonly in patients with hereditary non-polyposis colorectal cancer (HNPCC; Johnson et al., 2005). Mutations in β-Catenin interfere with the ability of the β-Catenin destruction complex to bind and phosphorylate β-Catenin and therefore deregulation of Wnt signalling occurs (Polakis, 2007). APC, AXIN, and CTNNB1 are the main genes that are mutated in genetic activation of many cancers. Mutations of some of the other components of the Wnt signalling pathway have also been shown to cause or contribute to the development of colorectal cancer. For example, mutation of TCF4, a transcription factor that is responsible for activation of Wnt targeted transcription, occurs in about half of colorectal cancer cases with microsatellite instability (Duval et al., 1999; Shimizu et al., 2002). 35  One of the consequences of constitutively active β-Catenin-dependent Wnt signalling in normal cells is the development of cancer. One of the downstream targets of the Wnt signalling pathway is c-MYC. c-MYC, a helix loop helix transcription factor (Soucek and Evan, 2010), is upregulated in a wide variety of cancers, including Burkitt’s Lymphoma, breast cancer, prostate cancer, gastrointestinal cancer, melanoma, multiple myeloma and myeloid leukemia (Nesbit et al., 1999). In experimentally induced murine epithelial hyperplasia, the loss of Apc activates c-Myc expression (Sansom et al., 2004), when Apc and c-Myc are both lost, the overgrowth caused by original Apc removal is inhibited despite high cellular β-Catenin levels (Sansom et al., 2007). c-MYC is also upregulated in colon cancer cell lines such as HT29, HCT116 and SW480 cells (He et al., 1998).  The expression of many genes is upregulated in colon cancer directly by the Wnt signalling pathway, such as cyclin D1 (CCND1; Tetsu and McCormick, 1999; Shtutman et al., 1999), peroxisome proliferator-activated receptor-δ (PPARD; He et al., 1999), c-JUN (Mann et al., 1999), matrix metalloproteinase-7 (MMP7; Brabletz et al., 1999; Crawford et al., 1999), CD44 (Wielenga et al., 1999), claudin-1 (CLDN1; Miwa et al., 2001), Survivin (BIRC5; Zhang et al., 2001), VEGF (Zhang et al., 2001), MET, a Receptor Tyrosine Kinase (Boon et al., 2002) and inhibitor of differentiation/DNA binding-2 (ID2; Rockman et al., 2001; Willert et al., 2002). Expression of some components of the Wnt signalling pathway is also upregulated in colorectal cancer, such as the TCF1, LEF1, and AXIN2. The overall effect of these changes is impaired apoptosis and differentiation, and the promotion of cell growth, proliferation, angiogenesis and metastasis. 36  Suppression of Wnt signalling in cancer cells can stop the growth of cancer cells. S100A4 is a calcium binding protein and a target of the Wnt signalling pathway (Sack et al., 2011). In colorectal cancer cells, S100A4 serves as a metastatic regulator.  When the transcription of S100A4 is inhibited in various colon cancer cell lines (HCT116, SW620, LS174T, SW480 and DLD-1 cells) by calcimycin (an inhibitor of β-Catenin-dependent Wnt signalling), cell growth, viability, migration, invasion and proliferation are all decreased (Sack et al., 2011). In NOD/SCID-IL2R- mice xenografted with human colorectal cancer HCT116 cells, calcimycin reduces the metastatic potential compared to that of the controls (Sack et al., 2011). LRP6 is a co-receptor required for β-Catenin-dependent Wnt signalling. Targeting LRP6 with niclosamide results in the inhibition of β-Catenin-dependent Wnt signalling, reduction in cell viability, and increased apoptosis in some types of breast and prostate cancer cells (Lu et al., 2011).  1.3.3 Evidence for Wnt and Folate Interactions in Colorectal Cancer There are some indications that folate and Wnt signalling pathway intersect. In several human colonic epithelial cell lines, folate depletion increases the expression of β-Catenin (CTNNB1) and APC (Crott et al., 2008). In contrast, folate depletion also upregulates the expression of p16, p21, and p53 tumour suppressor genes (Crott et al., 2008). These results are consistent with earlier findings showing an upregulated expression of CTNNB1 and downregulation of VEGF and p53 in four colorectal cell lines, including Caco2 cells (Novakovic et al., 2006). These observations suggest that folate deficiency affects the expression of genes involved in cell cycle regulation, D NA repair, apoptosis and angiogenesis in a cell-specific manner (Novakovic et al., 2006). In 37  C57BL/6J mice, folate deficiency (0 dietary folate, but no antibiotic administration) induces down regulation of Apc expression, upregulation and increased nuclear localization of β-Catenin, and upregulation of the downstream target cyclin D1 (Ccnd1) in the colon (Liu et al., 2007). Normal human fibroblasts cultured in 0 folate medium for 7 days show an altered distribution of cells in various phases of the cell cycle compared to the folate sufficient control, suggesting that proliferation and growth are inhibited by 0 folate conditions (Katula et al., 2007). Further, culturing in 0 folate conditions for 7, 10 or 14 days also upregulates the expression of WNT5a and WISP1 (Wnt1 inducible signalling pathway protein 1) and downregulates the expression of DKK1 (an inhibitor of Wnt signalling), which results in increased Wnt signalling activity (Katula et al., 2007).  A possible mechanism for the effect of folate on gene expression is through affecting DNA methylation. 5MTHF is a major methyl donor via SAM in the one-carbon metabolism pathway (Figure 1.2). One of the important functions of SAM is the methylation of CpG dinucleotides in DNA (Cedar, 1988). Of particular importance is the methylation of promoter regions of genes because an increase in methylation can lead to gene silencing by inhibiting the binding of transcription factors to the promoter region while decreased methylation could lead to gene activation (Cedar, 1988; Rountree et al., 2001). Aberrant DNA methylation is observed in a variety of cancers, in particular the hypermethylation of CpG dinucleotides in genes resulting in gene silencing has been intensely studied (Das and Singal, 2004).  For example, genes involved in apoptosis (e.g. DAPK and TMS1), cell cycle regulation (e.g. p16 and p15), and DNA repair (e.g. BRCA1 and MGMT) are frequently found to be hypermethylated in cancer (Das and Singal, 2004).  38  In colon cancer cells, hypermethylation induces silencing of SFRP (Suzuki et al., 2002), DKK (Aguilera et al., 2006) and WIF (He et al., 2005). As mentioned previously, these genes are all involved in inhibition of the β-Catenin-dependent Wnt signalling pathway. For example, hypermethylation of the SFRP1 promoter has been reported in various colorectal cancer cell lines, including RKO, Caco2, Colo205, DLD-1, HCT116, HT29, LoVo and SW480 (Suzuki et al., 2002). Stronger evidence linking hypermethylation to colorectal cancer is from human studies. In healthy individuals with no detected colorectal cancer, there is an absence of SFRP1 hypermethylation (Suzuki et al., 2002).  In contrast, SFRP1 is hypermethylated in primary colorectal cancer tissues (Suzuki et al., 2002). Similarly, other members of the SFRP family (SFRP 1-5) are also hypermethylated (Suzuki et al., 2002). To further understand the effects of hypermethylated SFRP in colorectal cancer cells, Suzuki et al. (2004) examined whether the correction of SFRP hypermethylation could downregulate the Wnt signalling pathway in colon cancer HCT116 cells, which possess mutated CTNNB1, and SW480 cells, which possess mutated APC. Equal expression of hemagglutinin-tagged SFRP1, SFRP2 and SFRP5 suppresses β-Catenin-mediated Wnt signalling in HCT116 and SW480 colorectal cancer cells (Suzuki et al., 2004). In these cell lines, stable overexpression of SFRP1, SFRP2 and SFRP5 results in a downregulation of MYC, a downstream target of Wnt signalling pathway (Suzuki et al., 2004). Similarly, the promoter region of SFRP1 is hypermethylated in 9 colorectal cancer cell lines examined (Aguilera et al., 2006). These studies have established that Wnt signalling can be constitutively upregulated in colorectal cancer cells when the promoters of SFRP genes are hypermethylated and silenced; however, the possible role of folate in the hypermethylation was not studied. 39  DKK1 is epigenetically silenced in several colorectal cancer cell lines (SW480, LS174T, HT29, LoVo and SW620), but not in normal lymphocytes and normal primary colorectal mucosal tissue (Aguilera et al., 2006). When the colorectal cancer cells with low-level expression of DKK1 are treated with a demethylation agent, DKK1 expression is restored (Aguilera et al., 2006). Further, DKK1 silencing appears to be correlated to colorectal cancer stage (Aguilera et al., 2006). In cell lines derived from advanced or late stage colorectal cancer, the expression of DKK1 is silenced; however in cell lines derived from earlier stage colorectal cancers, the promoter region for DKK1 is not hypermethylated, and therefore not silenced (Aguilera et al., 2006). This same relationship is also observed in primary tumours: DKK1 is hypermethylated in the most clinically advanced tumours (Aguilera et al., 2006). This is in contrast to the methylation of SFRP1, which is independent of cell lines and the cancer stage of the primary tissue from which the cells are derived (Aguilera et al., 2006). Also in contrast to SFRP1, restoration of DKK1 expression does not inhibit basal Wnt signalling in either late stage- or early stage-derived colorectal cancer cell lines (Aguilera et al., 2006). However, restoration of DKK1 expression inhibits the growth of DLD-1 colorectal cancer cell colonies (Aguilera et al., 2006).  Similarly, tumor size is reduced in mice xenografted with DLD-1 cells transfected with DKK1 compared to the control (Aguilera et al., 2006). WIF1 is another gene where methylation status and expression have been altered in colorectal cancer cells (Aguilera et al., 2006; He et al., 2005). WIF1 is expressed in normal colon cells, but its expression is much lower in SW480 colorectal cancer cells and non-existant in HCT116 colorectal cancer cells (He et al., 2005). Methylation-specific PCR showed that WIF1 is not methylated in normal colon cells, but is hypermethylated in 40  both SW480 and HCT116 colorectal cancer cells (He et al., 2005). In SW480 colorectal cancer cells, restoration of WIF1 expression or inhibition of WNT1 with siRNA induces apoptosis (He et al., 2005). In primary advanced-staged colorectal cancer tissues, expression of WIF1 is either downregulated or absent in 5 out of 7 individuals (He et al., 2005). This status of WIF1 expression correlates with the methylation status of the WIF1 promoter (He et al., 2005). Epigenetic silencing of SFRP, WIF1 and DKK1 suggests that upstream Wnt signalling is important in carcinogenesis. In the case of SFRP and WIF1, restoring expression of these genes in colorectal cancer cells inhibits the Wnt/β-Catenin signalling pathway. Both SFRP and WIF1 exert their inhibitory functions on the Wnt signalling pathway by directly binding to Wnt proteins. Restoring expression of DKK1 in colorectal cancer cells did not inhibit Wnt signalling. However in DLD-1 colorectal cancer cells, tumour growth is inhibited when DKK1 expression is restored, suggesting that anti-growth effect of DKK-1 is independent of the Wnt signalling pathway.  Expression of several other genes encoding proteins in the Wnt signalling pathway is regulated by methylation. For example, WNT5A (a tumour suppressor) is hypermethylated in 5 out of 6 colorectal cancer cell lines, including Caco2 cells, and unmethylated in a normal colon cells (Ying et al., 2008). Treatment of the colorectal cancer cells exhibiting hypermethylated WNT5A promoters with a demethylating agent (5-Aza-2’-deoxycytidine) restores WNT5A expression (Ying et al., 2008). Hypermethylation in the WNT5A promoter is present in 48% of primary colorectal cancer samples, compared to only 13% of normal primary colon tissue samples (Ying et al., 2008). WNT5A is not expressed in HCT116 colon cancer cells. Transfecting HCT116 cells with WNT5A significantly inhibits tumour colony formation compared to the 41  control, while both β-Catenin protein levels and luciferase reporter activity and  mRNA levels of CCND1, a downstream target of Wnt/β-Catenin signalling, are significantly reduced  (Ying et al., 2008). Increased global DNA methylation has been shown in several clinical trials in patients with colorectal adenomas when they are treated with daily folate supplements (400 µg, 5 mg and 10 mg FA/d; Kim, 2005). In one study conducted on patients with colonic adenomas, supplementation with 5 mg/d FA promotes global DNA methylation in rectal mucosa if the patient has 1 adenoma (Cravo et al., 1998). Cravo et al. (1994) reported that cancerous tissues from patients with colorectal carcinomas are hypomethylated compared to normal colonic tissue and adenoma tissue, but both patients with adenomas and carcinomas exhibit increased global DNA methylation after FA treatment (10 mg/d). In a clinical intervention trial, colorectal cancer patients with adenomas were randomized to receive either FA supplements (5 mg/d) or a placebo for 1 year with the outcomes being global DNA methylation of rectal mucosa and p53 strand breaks assessed at 6 months and 1 year (Kim et al., 2001). Patients receiving FA have increased global DNA methylation and decreased p53 strand breaks, while adenoma recurrence is not affected after 1 year (Kim et al., 2001). In the most recent study looking at global DNA methylation in patients with colorectal adenomas, FA supplementation  (400 µg/d) was non-significantly associated with higher or greater global DNA methylation in the rectal mucosa by 25% (95% CI, 0.11 – 39%, p=0.09) as compared to the placebo group (Pufulete et al., 2005). These studies all have similar results with differing supplementation levels of FA: increase in global DNA methylation. The authors state that global DNA hypomethylation may lead to the development of colorectal cancer; 42  however these studies only looked at people who already had cancer. The consequences of increasing global DNA methylation in patients who do not already have cancer are not established. As in vitro evidence has shown, hypermethylation of certain genes is observed in cancer cells lines and primary culture. It is not known whether daily folate supplementation could increase promoter hypermethylation of these genes. The link between folate, colorectal cancer and perhaps Wnt signalling is widely hypothesized to be attributed to the role of folate in DNA methylation; however there are other studies showing that nuclear localization of β-Catenin is affected by folate as well. Translocalization of β-Catenin from the cytoplasm to the nucleus is essential for Wnt signalling pathway activity. In NIH3T3 cells, folate deficiency was shown to increase β-Catenin nuclear localization (Morillon II, 2008). However, β-Catenin protein levels do not differ significantly between the folate sufficient and deficient cells (Morillon II, 2008). This observation supports an earlier observation that folate supplementation in patients with colorectal adenomas decreases β-Catenin localization in the nucleus of rectal mucosa cells (Jaszewski et al., 2004).   1.4 Summary FA fortification may have adverse effects in relation to colorectal cancer in certain groups of the population. There is a need for research to establish whether FA has a differential effect between normal cells and cancerous cells compared to 5MTHF. Introduction of FA fortification in several nations has been linked to a rise in colon cancer incidence. However, this issue is controversial because studies also point to folate deficiency being a risk factor for cancer progression. Most cases of colorectal cancer 43  have genetic mutations that result in constitutive upregulation of the Wnt signalling pathway, leading to increased proliferation, vascularization, metastatic potential and decreased apoptosis. There is evidence that folate and Wnt signalling are linked: inhibitors of Wnt signalling are epigenetically silenced in many colorectal cancer cases and β-Catenin nuclear translocation is increased during folate deficiency.   1.5 Hypothesis  The hypothesis for my thesis research is that folic acid will affect the proliferation and folate transporter expression in colon cancer cells differently than L-5-methyltetrahydrofolate. In addition, the forms of folate, reduced versus oxidized, will also differentially affect the activity of the Wnt signaling pathway.   1.6 Overall Objective and Specific Aims The objective of my research is to investigate the effect of folic acid and L-5-methyltetrahydrofolate on the proliferation, expression of selected folate transporters, and the activity of the Wnt signalling pathway in human colorectal adenocarcinoma Caco2 cells. The specific aims of my thesis research are: 1) To establish the effect of the form and concentration of folate on the proliferation and viability of Caco2 cells. 2) To assess the effect of the form and concentration of folate on the expression of folate transporters in Caco2 cells. 44  3) To investigate the effect of the form and concentration of folate on the intracellular translocation of β-Catenin, a key molecule in the Wnt signalling pathway, in Caco2 cells.                     45  Table 1.1: Food Sources of Folate (Canadian Nutrient File, 2010) Food Source Serving Size Folate (µg) Asparagus, cooked 4 spears 80-88 Baby soybeans, cooked 125 mL 106-255 Bagel, plain ½ bagel (44.5 g) 101 Beans, cranberry/roman, cooked 175 mL 271 Beans (kidney, great northern), cooked 175 mL 157-170 Beans (mung, adzuki), cooked 175 mL 234-238 Beans (navy, black, small white), cooked 175 mL 181-190 Beans (pink, pinto), cooked 175 mL 210-218 Bread, white 1 slice (35 g) 60 Bread, whole wheat 1 slice (35 g) 18 Broccoli, cooked 125 mL 89 Lentils, cooked 175 mL 265 Liver, (beef, pork), cooked 75 g 122-195 Liver (lamb, veal), cooked 75 g 262-300 Liver (turkey, chicken) cooked 75 g 420-518 Okra, frozen, cooked 125 mL 142 Orange juice 125 mL 58 Pasta, egg noodles, enriched, cooked 125 mL 138 Pasta, white, enriched, cooked 125 mL 83-113 Peas (chickpeas, black-eyed/cowpeas/adzuki),cooked 175 mL 180-263 Peas, pigeon, cooked 175 mL 138 Spinach, cooked 125 mL 121-139 Turnip greens or collards, cooked 125 mL 68-93 *According to the Canadian Food Inspection Agency, a food must contain ≥5% of the RDI of a vitamin or mineral to be labelled a source (information updated 2014-03-25).   46   A)  B)  Figure 1.1: The structures of A) folic acid (FA) and B) 5-methyltetrahydrofolate (5MTHF).         47   Figure 1.2: The folate metabolism pathway (Zhao et al., 2009. Adopted with modifications by permission from Expert Reviews in Molecular Medicine). Abbreviations: AICAR (phosphoribosylaminoimidazolecarboxamide transformylase), B12 (Vitamin B12), GAR (phosphoribosylglycinamide transformylase), SHMT (serine hydroxymethyltransferase), THF (tetrahydrofolate).                 48  A) B) Figure 1.3: Schematic of canonical Wnt/β-Catenin-dependent Wnt signalling. A) No Wnt stimulation and B) with Wnt stimulation. Abbreviations: APC (Adenomatous polyposis coli), β-cat (β-Catenin), CK1 (Casein kinase 1), DSH (Dishevelled), FZD (Frizzled), GSK3 (Glycogen synthase kinase 3), LRP5/6 (Lipoprotein receptor-related protein 5/6), TCF/LEF (T cell factor/Lymphoid enhancer-binding factor), Wnt (Wingless type proteins). 49    Figure 1.4:  Colorectal Cancer rates pre- and post-fortification in the U.S. and Canada (Mason et al., 2007. Reproduced with permission from Cancer Epidemiology, Biomarkers & Prevention)   50  CHAPTER 2 Effects of Short-term Supplementation of Folic Acid and L-5-methyltetrahydofolate on Proliferation, Viability and the Expression of Folate Transporters  in Human Colorectal Adenocarcinoma Caco2 Cells  2.1 Introduction An unexplained increase in colorectal cancer incidence has been observed in Canada, the United Sates and Chile post-folic acid fortification (Mason et al., 2007; Hirsch et al., 2009). These observations, as well as reported rises in all cancer incidence and mortality observed in patients who received FA and vitamin B12 supplements (Ebbing et al., 2009), give cause for the concern that FA fortification may be harmful for certain sub-populations.  Dietary folate has been shown to affect the expression of PCFT, FR, and RFC, a group of major folate importers, and BCRP (Zhao et al., 2009), a folate exporter in the small intestine, colon, liver, ovary and placenta (Maliepaard et al., 2001).  These receptors transport folate through various mechanisms.  For example, PCFT is a folate-proton symporter (Qiu et al., 2006) while RFC is an anion exchanger (Goldman, 1971).  In contrast, FRs transport folate through receptor-mediated endocytosis (Kamen et al., 1988).  FA is an oxidized form of folate whereas 5MTHF is a reduced form of the vitamin.  This difference in their chemical structure influences their binding affinity towards these folate transporters.  For example, PCFT, which transports folate best at an acidic pH, has a Km of 0.53, 0.83, and 2.01 µM for 5MTHF, FA, and methotrexate, respectively, at pH 5.5 (Qiu et al., 2006), indicating that PCFT has a higher affinity for 5MTHF than FA and methotrexate. The difference in affinities becomes more apparent if 51  the pH is increased to 6.5, where Km for 5MTHF, FA, and methotrexate is 0.78, 2.99, and 8.07 µM, respectively (Qiu et al., 2006). 5MTHF and other reduced folates are transported by RFC with a higher affinity (Km = 1 – 3 µM) than FA (Km = 200 – 400 µM; Jansen et al., 1997). In contrast, FRα has a higher affinity for FA (KD = 1 pM) than 5MTHF (KD ≈ 1 nM; Kamen and Smith, 2004).  Clearly, folates, such as 5MTHF, and FA are likely transported by these transporters with different efficiencies. The Wnt signalling pathway is important for many developmental processes in the body (Logan and Nusse, 2004). Aberrant Wnt signaling has been linked to the development and progression of many diseases, especially cancer (Logan and Nusse, 2004). In sporadic or hereditary colorectal cancer, Wnt signalling is often aberrant. Therefore folate could affect the Wnt signaling pathway, possibly through gene promoter methylation (Suzuki et al., 2004). In fact, it has been shown that a number of Wnt signaling pathway inhibiters are hypermethylated in primary colorectal cancer samples and colon cancer cell lines (Suzuki et al., 2002; Suzuki et al., 2004; He et al., 2005; Aguilera et al., 2006; Ying et al., 2008). It has previously been shown that folate deficiency affects β-Catenin (an effector of Wnt signalling) localization into the nucleus in NIH3T3 cells (Morillon II, 2008). β-Catenin movement to the nucleus is essential for the activity of the Wnt signaling pathway. Therefore folate could be involved in the Wnt signaling pathway over and above its role in DNA methylation.  I hypothesize that folic acid will affect the proliferation and folate transporter expression in colon cancer cells differently than L-5-methyltetrahydrofolate. In addition, the forms of folate, reduced versus oxidized, will also affect the activity of the Wnt signaling pathway differently. The specific aims of my thesis research are: 1) to establish 52  the effect of the form and concentration of folate on the proliferation and viability of Caco2 cells; 2) to assess the effect of the form and concentration of folate on the expression of folate transporters in Caco2 cells; and 3) to explore the effect of the form and concentration of folate on the translocation of β-Catenin, a key molecule in the Wnt signaling pathway, in Caco2 cells.  2.2 Materials and Methods 2.2.1 Cell Culture System and Folate Treatments  Human colorectal adenocarcinoma Caco2 cells (HTB-37; ATCC, Manassas, VA), passage number 20 – 30, were selected as the cell culture system for this research.  The Caco2 cells were routinely maintained in Dulbecco’s modified essential medium (DMEM; Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (FBS), sodium pyruvate (1 mM), penicillin (100 U/mL) and streptomycin (100 µg/mL) in a humidified atmosphere at 37°C, 10% CO2.   In preparation for  the 5 day experiments, cells were initially cultured in a folate-free RPMI 1640 medium (ff-RPMI 1640; Gibco, Grand Island, NY) containing 10% dialyzed FBS (dFBS; Gibco, Grand Island, NY), sodium pyruvate (1 mM), pencillin (100U/mL), and streptomycin (100 µg/mL) for 5 days in a humidified atmosphere at 37°C, 5% CO2. To examine the effect of folate on cell proliferation and viability, FA (Sigma-Aldrich, St. Louis, MO) or 5MTHF (Calcium salt; Merck, Whitehouse Station, NJ) was added to the treatment medium at 0, 0.9, 2.3, or 3.4 µM.  53  These supplementation levels were designed to deliver 400, 1,000 and 1,500 µg folate/L (0.9, 2.3, and 3.4 µM folate, respectively) to mimic adequate folate intake, and medium and high folate supplementations, respectively.  In order to prevent oxidation of 5MTHF, sodium ascorbate (5%; Sigma-Aldrich, St. Louis, MO) was added to the 5MTHF stock solutions (1 mM).  The stock solution was sterilized with a 0.22 µm syringe filter and stored at -20oC until use for up to 1 month.  The treatment duration of 3 days was initially chosen to investigate the acute response to folate supplementation. Akoglu et al. (2001, 2004) have shown effects of different folates (1.4 – 22.6 µM) on the growth and viability of Caco2 cells after 24 hours and 48 hours.   2.2.2 Assessment of Cell Viability and Proliferation  Caco2 cells were sub-cultured in ff-RPMI 1640 supplemented with 0 (5% sodium ascorbate), 0.9, 2.3 or 3.4 µM FA or 5MTHF for 3 or 5 days in a 96-well plate. For the 5 day experiment, Caco2 cells were initially cultured in ff-RPMI 1640 for 5 days in a 10 cm culture dish. The initial seeding density was 5,000 and 2,000 cells/well for the 3 and 5 day experiments, respectively. At the end of the treatment duration, cells were 60 – 80 % confluent. Cell viability following folate treatment was assessed using the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay (Roche, Indianapolis, IN) according to the manufacturer’s instruction.  The WST-1 assay measures the reduction of WST-1, a water-soluble tetrazolium salt, to formazan, a water-soluble dye, by mitochondrial succinate-tetrazolium reductase present in viable cells 54  (Ngamwongsatit et al., 2008).  Briefly, WST-1 (20 µL) was added to each well and mixed by placing the plate on a Thermomixer (400 rpm, 55°C, 1 min; Thermomixer R, Eppendorf, Mississauga, ON) followed by incubation at 37°C, 5% CO2 for 4 h. Subsequently, the reaction mixture was mixed again with the Thermomixer under the same conditions described above.  The formazan produced was measured using a microplate reader (SpectraMax Plus, Molecular Devices, Sunnyvale, CA) at 450 nm with a reference wavelength at 690 nm.  To assess cell proliferation, cells were cultured and treated with FA or 5MTHF as described above.  At the end of the 3 or 5 day treatment period (cells were 70 – 80% confluent), the cells were subjected to the 5-bromo-2’-deoxyuridine (BrdU) colourimetric assay (Roche, Indianapolis, IN) according to the manufacturer’s instruction. The intensity of the color developed was determined at 405 nm using the same microplate reader described above with a reference wavelength of 490 nm. The BrdU assay measures the incorporation of BrdU (a nucleotide analogue) into newly synthesized DNA and therefore, only cells undergoing active DNA synthesis will be labelled with BrdU (Terry and White, 2006).   2.2.3 Cell Cycle Analysis   Caco2 cells were cultured in ff-RPMI-1640 medium at an initial seeding density of 500,000 and 250,000 cells/10 cm plate (3 and 5 day experiments, respectively) and treated with either FA or 5MTHF at the concentrations described above for 3 or 5 days. For the 5 day experiement, cells were first cultured in ff-RPMI 1640 for 5 days. At the end of the treatment period, the cells were 70 – 80% confluent. Subsequently, the cells 55  were incubated with BrdU (Sigma-Aldrich, St. Louis, MO) at a final concentration of 10 µM (10 µL of BrdU stock solution (1 mM) / mL of culture medium) for 1 h at 37°C, 5% CO2. The cells were protected from light after addition of BrdU until the completion of the assay. At the end of the incubation period, cells were subjected to cell cycle analysis using a flow cytometric assay (Terry & White, 2006) with modifications. Briefly, cells were harvested with 2 mL 0.25% Trypsin-EDTA (Gibco, Grand Island, NY), neutralized with an equal volume of ff-RPMI 1640 medium. Cells were then counted by diluting 0.1 mL of cell suspension in 9.9 mL of IsoFlow™ diluent (Beckman-Coulter, Indianapolis, IN) using a particle counter (Z1 Coulter® Particle Counter, Beckman-Coulter, Indianapolis, IN), and pelleted by centrifugation (400 x g, 4 min, 20°C). The supernatant was then aspirated and cells were fixed by slowly adding cold PBS at 0.8 mL/2 million cells and subsequently trickling in 100% ethanol (-20°C) at 1.2 mL/2 million cells while vortexing to a final concentration of 1 million cells/mL.  Cells were then left at 4°C overnight.  After fixing overnight, the cells were resuspended by vortexing and collected by centrifugation (400 x g, 4 min, 20°C). After removing the supernatant, HCl (2 N; 3 mL) was added to each tube while vortexing followed by incubation at 37°C for 30 min.  During the incubation, the cells were mixed by vortexing every 10 min (twice). After the incubation, sodium borate (0.1 M; 6 mL) was added to each tube while vortexing and pelleted by centrifugation (400 x g, 4 min, 20°C). The cell pellet was slowly suspended in 6 mL PBTB (PBS containing 0.5% Tween-20 and 0.5% bovine serum albumin (BSA)) while vortexing followed by centrifugation (400 x g, 4 min, 20°C).  The cells were subsequently incubated with anti-BrdU mAb (sc-20045, Santa Cruz Biotechnology, Santa 56  Cruz, CA) at 1:100 dilution (0.3 mL for up to 20 million cells in PBT (PBS containing 0.5% Tween-20) for 60 min at room temperature in the dark.  After incubation with the primary antibody, the cells were rinsed with 3 mL PBTB while vortexing, pelleted by centrifugation (400 x g, 4 min, 20°C), and incubated with goat-anti mouse IgG1-FITC antibody (sc-2078, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100 dilution (0.2 mL for up to 20 million cells in PBTG (PBTB containing 1% normal goat serum)) for 45 min at room temperature in the dark. At the end of incubation period, 3 mL PBTB was added to the incubation solution and counted using the same particle counter described above.  The volume of the cell suspension was adjusted to yield 1 million cells/tube. The labelled cells were either analyzed on the same day or stored at 4°C overnight in the dark and analyzed the next day.   Before analysis, the cells were collected by centrifugation (400 x g, 4 min, 20°C) and stained with propidium iodide (PI; Sigma-Aldrich, St. Louis, MO) in PBTB containing RNase A.  The final concentration of PI and RNase A was 10 and 20 µg/1 million cells/mL, respectively. After incubating the cells for 30 min in the dark at room temperature, the cells were analyzed using a flow cytometer (FACScalibur; Beckton-Dickinson and Company, Franklin Lakes, NJ) with excitation at 488 nm (5-W argon-ion laser operating at 200 mW). BrdU (FITC, green fluorescence) was measured after blocking incident laser light using a logarithmic amplifier with a 530 nm short-pass filter. Linear DNA content was measured using PI (red fluorescence) with a 610 nm long-pass filter. Data was collected using Cell Quest (Beckton-Dickinson and Company, Franklin Lakes, NJ) and analyzed using FlowJo software (Version 7.6.5, Tree Star Inc., Ashland, OR). 57  2.2.4 Whole Cell Lysate Preparation and Western Blot Analysis  Caco2 cells were cultured in ff-RPMI-1640 for 5 days with an initial seeding density of 250,000 cells/10 cm dish and subsequently treated with FA or 5MTHF for 5 days as described above. At harvest, cells were 70 – 80% confluent.  To prepare the whole cell lysate, cells were rinsed with warm PBS (pH 7.4; 37°C) and harvested with 0.25% Trypsin-EDTA followed by neutralization with equal volume of the culture medium containing FBS.  The harvested cells were pelleted with centrifugation (700 rpm, 5 min, 4°C). The cell pellet was resuspended in PBS and counted as described above. The cells were then repelleted with centrifugation (700 rpm, 5 min, 4°C), resuspended in 1 mL PBS and subsequently transferred to a microcentrifuge tube.  After pelleting (14,000 x g, 5 min, 4°C), the cells were suspended in whole cell lysis buffer (pH 7.4; 50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulphonyl-fluoride (PMSF)) in the presence of 10% protease inhibitor cocktail (AEBSF, 104 mM; Aprotinin, 80 μM; Bestatin, 4 mM; E-64, 1.4 mM; Leupeptin, 2 mM; Pepstatin A, 1.5mM; P8340; Sigma-Aldrich, St. Louis, MO) at 30 µL whole cell lysis buffer/106 cells.  The cell suspension was incubated on ice for 30 min. After the incubation, the lysis buffer-cell mixtures were centrifuged at 14,000 x g for 10 min at 4°C and the supernatant was transferred to a new microcentrifuge tube.  Total cellular protein was quantified using BioRad DC System (BioRad, Hercules, CA, USA).  Total cellular protein (50 µg/well) was prepared by diluting the volume to 10 µL with whole cell lysis buffer (without protease inhibitors or PMSF) and adding 10 µL 2X loading buffer (0.125 M Tris (pH 6.8), 5% SDS, 3.73 mM bromophenol blue, 10% 58  glycerol, ddH2O; For RFC and PCFT, 2 µL dithiothreitol was added to 8 µL 2X loading buffer, for FRα and BCRP, no dithiothreitol was used), for a total of 20 µL/well). Protein and loading buffer were then boiled for 5 minutes and then electrophoretically separated on a 10% (RFC1, FR-α, and PCFT) or 12% (BCRP) SDS-PAGE at 175 V until the dye front reached the bottom of the gel (approximately 55 min).  The proteins were electrotransferred at constant current (200 mA) to a PVDF membrane (0.2 µm; Immobilon®-PSQ; Millipore, Billerica, 3 h) using a Tris-glycine transfer buffer (25 mM tris, 0.192 M glycine, 20% methanol and ddH2O; stored for maximum 1 week at 4°C). After transfer, the membrane was washed with TBS (50 mM Tris and 150 mM NaCl) twice (10 min/wash) and blocked with 2.5% skim milk powder in TTBS buffer (50 mM Tris, 150 mM NaCl, 0.05% Tween 20) for 75 min.  To detect the targets, the membrane was incubated with the appropriate primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in 2.5% skim milk powder in TTBS buffer overnight at appropriate dilution (Table 2.1) followed by washing with TTBS (3 X; 10 min/wash). Subsequently, the membrane was incubated with the appropriate secondary antibody in 1% skim milk powder in TTBS buffer for 1 h and washed with TTBS (3 X, 10 min/wash).  The primary antibodies and their corresponding secondary antibodies and the dilution factors are listed in Table 2.1. Presence of the target proteins was detected using the enhanced chemiluminescence kit (ECL; Thermo Scientific, Rockford, IL) and visualized with chemiluminescence scientific imaging film (Kodak BioMax Light film, Sigma-Aldrich, St. Louis, MO) or using BioRad Chemidoc (BioRad, Hercules, CA). Band intensity was quantified with ImageJ software (version 1.45s, US NIH, Bethesda, MD).   59  2.2.5 Cellular localization of -catenin   Caco2 cells were initially grown in ff-RPMI 1640 for 5 days and then subcultured onto a glass coverslip (12 x 12 x 1.5 mm), which was placed in a well in a 24-well plate at an initial seeding density of 20,000 cells/well, and treated with FA or 5MTHF as described above. At seeding, the cells were well dispersed, and not confluent. After the treatment period, the cells were 70 – 80% confluent. After 5 days of treatment with FA or 5MTHF, the cells were rinsed with PBS and fixed with 100% methanol for 20 min at -20°C. After fixation, the cells were rinsed with ice cold PBS twice.  The cells were then permeabilized with 0.1% Triton X-100 in PBS for 10 min followed by washing with PBS three times (5 min/wash). Subsequently, cells were incubated with 0.5% BSA in PBT for 30 min to block unspecific binding of antibodies and labelled with rabbit anti-β-Catenin polyclonal antibody (AHO0462; Invitrogen, Grand Island, NY) at 15 µg antibody/mL hybridization solution (0.5% BSA in PBT) at room temperature for 1 h. The cells were then washed with PBS three times (5 min/wash). After washing, the cells were incubated with Alexa Fluor® 488 goat anti-rabbit IgG (A11008; Invitrogen, Grand Island, NY) at 5 µg antibody/mL hybridization solution at room temperature, dark, for 1 h followed by 3 PBS washes (5 min/wash).  Finally, the cells were counterstained with DAPI and mounted on a glass microscope slide in the same step using ProLong® Gold Antifade Reagent with DAPI (P-36931; Invitrogen, Grand Island, NY).  Three drops of the reagent were placed on a glass microscope slide and a coverslip was placed on top of each drop with the side that cells grown on faced down. After curing overnight at room temperature in the dark, each 60  coverslip was sealed with clear nail polish and left to dry at room temperature before examining samples. Samples were visualized with a Fluoview FV10i confocal microscope (Olympus, Center Valley, PA). Images were taken with the 60 x objective, zoomed in 2 x for a total of magnification of 120 x (Olympus Fluoview FV1000, version 2.1c; Olympus, Center Valley, PA). Image resolution is 512 x 512 pixels and aperture was x 1.0. β-Catenin localization was represented by the fluorescence of Alexa fluor 488® excited at 473 nm and counterstaining with DAPI was excited at 405 nm. Images were taken as z stacks with each slice 0.819 µm thick. Images were analyzed using Volocity 3D Image Analysis Software (Version 6.1.1; PerkinElmer, Waltham, MA).  2.2.6 Statistical Analysis:   All data was analyzed with two-way ANOVA. The ANOVA showed that there was a significant effect of the form of folate on 3 day cell viability and both form and concentration of folate on 3 day cell proliferation; but there was no significant effect of the concentration and interaction between the form and concentration of folate tested on all other end points. One-way ANOVA was performed followed by Tukey’s Honestly Significant Difference Test for determining the effects among the folate concentrations within the same form of folate and T-test for determining the effects between FA and 5MTHF folate at the same concentration (p < 0.05; SPSS Statistics 22, IBM, Armonk, NY).     61  2.3 Results 2.3.1 Cell Viability 3-day folate treatment.  Cell viability was not affected by FA and 5MTHF at the concentrations tested compared to the control (Figure 2.1).  Cell viability was 15.8% higher in cells treated with 0.9 µM of FA than that in cells treated with 5MTHF at the same concentration (p = 0.044; Figure 2.1).  Cell viability did not differ between FA and 5MTHF treatment at 2.3 and 3.4 µM (Figure 2.1).   5-day folate treatment.   Cell viability was not affected by treating the cells with FA and 5MTHF at 0.9, 2.3, and 3.4 M compared to the control (Figure 2.2).  There was no difference in cell viability between the cells treated with FA or 5MTHF (Figure 2.2).  2.3.2 Cell Proliferation 3-day folate treatment.   Cell proliferation was increased by 11 (p = 0.018), 12 (p = 0.001), and 21% (p = 0.021) in Caco2 cells treated with 0.9, 2.3, and 3.4 µM of FA, respectively, for 3 days compared to that in cells treated with same concentrations of 5MTHF (Figure 2.3). Cell proliferation was 23% higher in cells treated with 3.4 µM FA compared to the control (p = 0.008), but was not affected by treating the cells with 0.9 and 2.3 M of FA (Figure 2.3). Cell proliferation in Caco2 cells was also not affected by 5MTHF treatment at all three concentrations tested (Figure 2.3). 5-day folate treatment. Cell proliferation was not affected by treating Caco2 cells with 0.9 and 3.4 µM of FA or 5MTHF at all three concentrations tested for 5 days compared to the control (Figure 2.4). There was also no effect of cell proliferation in cells treated with FA or 5MTHF for 5 days (Figure 2.4).  62  2.3.3 Cell Cycle Analysis 3-day folate treatment. Results of cell cycle analysis are shown in Figure A.1 and summarised in Table 2.2.  After 3-day folate treatment, there was no significant difference in the proportion of cells in pre-G1 phase (Table 2.2). Over half (52.5%) of the control cells were in the G1 phase while 34.1 and 8.6% of the cells had progressed into the S and G2/M phases, respectively (Table 2.2). Treating the cells with 0.9 and 2.3 µM of FA or 5MTHF had no effect on the cell cycle progression compared to the control.  However, treating the cells with 3.4 M of FA resulted in an 8.4 and 7.9% reduction in the proportion of cells in the G1 phase compared to the control and cells treated with 5MTHF at the same concentration, respectively (Table 2.2).  Furthermore, treating the cells with 3.4 M of FA also resulted in a 15.9% increase in the proportion of cells in the S phase compared to the cells treated with 5MTHF at the same concentration, while the proportion of cells in the G2/M phases remained the same between these two groups of cells. Treating the cells with 3.4 M of 5MTHF had no effect on the proportion of cells in the G2/M phase compared to the cells treated with 3.4 µM of FA. 5-day folate treatment.  Results of the cell cycle analysis are shown in Figure A.2 and summarised in Table 2.3.  Treating the cells with FA or 5MTHF at 0.9, 2.3, and 3.4 M for 5 days had no effect on cell cycle progression compared to the control, except in the pre-G1 phase (Table 2.3). At 3.4 µM of 5MTHF, there was a 49.3% increase in the proportion of cells in the pre-G1 phase compared to the cells treated with FA at the same concentration. Treating the cells with 3.4 M of FA and 5MTHF increased the proportion of cells in the pre-G1 phase by 3.6 and 5.3 times, respectively, compared to the control.  Treating the cells with FA and 5MTHF at 0.9 µM resulted in an 8% increase in the 63  proportion of cells in the G1 phase compared to the control. In contrast, treating the cells with FA and 5MTHF at 0.9 µM resulted in 19.6 and 17.4% reduction in the proportion of cells in the S phase compared to the control, respectively. At 3.4 µM, 5MTHF treatment resulted in a 26.6% decrease in the proportion of cells in G2/M compared to the control. Comparing the data from 3 day and 5 day folate treatment showed a large difference in cell cycle analysis. The proportion of cells in the G1 phase is much higher in Caco2 cells treated with either FA or 5MTHF for 5 days (61.9 – 67.3 %; Table 2.3) compared to the cells treated with the same treatments for 3 days (48.1 – 52.5 %; Table 2.2). On day 5, the folate treated groups had percentages of cells in the S phase ranging from 18 – 22.4% (Table 2.3). The percentage of cells in the S phase of the 3 day folate-treated Caco2 cells was 30.8 – 35.7% (Table 2.2).   2.3.4 Folate Transporters Treating Caco2 cells with FA and 5MTHF at 0.9, 2.3, and 3.4 µM had no effect on the abundance of RFC (Figure 2.5), FRα (Figure 2.6), and BCRP (Figure 2.7) compared to the control. Due to very low abundance of the PCFT in non-confluent cells, there was no detectable signal in all treatment groups (Results are not shown).   2.3.5 Nuclear Localization of β-Catenin  The nuclear localization of β-Catenin was not affected by folate treatment, regardless of the form and concentrations of folate that Caco2 cells were exposed to (Figures 2.8 and 2.9).   64  2.4 Discussion 2.4.1 Folate Treatment Did Not Affect Caco2 Cell Viability and Proliferation It has been well documented that folate deficiency decreases cell viability and proliferation. Folate deficiency affects DNA stability through its role as a carbon donor in the synthesis of thymine from uracil (Duthie et al., 2002). In times of cellular folate deficiency, the metabolism of one-carbon units is reduced. In theory, treating Caco2 cells treated with FA or 5MTHF should have increased cell viability and proliferation compared to the cells cultured in the control medium, which contained very small levels of folate due to the dFBS. In this study, there was no difference in cell viability between the control and the FA or 5MTHF treatment groups after 3 days (Figure 2.1). In terms of cell proliferation, treating the cells with FA at 3.4 µM significantly increased DNA synthesis compared to the control (Figure 2.3); however treating the cells with FA at lower concentrations had no effect on DNA synthesis compared to the control. Further, treating cells with 5MTHF at all three concentrations tested had no effect on DNA synthesis.  This lack of consistency in folate-induced promotion of DNA synthesis suggests that three days may not be sufficiently long to deplete cellular folate level in the control cells to suppress DNA synthesis in these cells.  Cell proliferation was also investigated with cell cycle analysis. After 3 days folate treatment, compared to the control, there were no differences in the proportion of cells in each phase of the cell cycle (Table 2.2). This finding was consistent with the cell proliferation and cell viability results: after 3 days, there is no effect of folate treatment on the growth of Caco2 cells compared to the control. 65  Since 3 day folate treatment did not elicit an effect, a longer treatment period of 5 days was investigated. Caco2 cells were first grown in folate free medium for 5 days, followed by a 5 day treatment with either FA or 5MTHF. This 5 day depletion was done to “reset” the cells so their response to FA and 5MTHF could be better observed. In studies with Caco2 and other colon cancer cells lines, 20 days culture in folate free medium depleted the intracellular folate to 98-99% of control (Novakovic et al., 2006). Following 5 days culture in 0 folate and 5 days treatment with FA or 5MTHF, there was no difference in cell viability or cell proliferation compared to the control cells (Figure 2.2, Figure 2.4). Cell cycle analysis, on the other hand, revealed 3.6 and 5.3-fold differences in the proportion of cells in the pre-G1 between the treated cells (3.4 µM FA and 5MTHF, respectively) and the control (Table 2.3).   After 5 days treatment, Caco2 cells treated with 3.4 µM FA or 5MTHF the proportion of cells in pre-G1 phase were much higher than the control (Table 2.3; Figure A.2). Caco2 cells treated with 5MTHF had 20.3% of cells in pre-G1 whereas FA treated cells had 13.6% of cells in pre-G1 phase, compared to the control (3.8%; Table 2.3). A possible explanation for the spikes in the proportion of cells in the pre-G1 phase was that the Caco2 cells were becoming confluent. As Caco2 cells approach confluency the proportion of cells in G1 increases while the proportion of cells in the S phase decreases (Ding et al., 1998). As Caco2 cells grow towards confluence they begin to differentiate into a cell with a small intestinal enterocyte phenotype and they start to exhibit contact inhibition and subsequently go into cell cycle arrest (Stierum et al., 2003).  Though the proportion of cells in the pre-G1 phase was higher after treatment with 3.4 µM FA or 5MTHF, the proportion of cells in G1 phase did not change after 66  folate treatment (Table 2.3). However, treatment with 3.4 µM 5MTHF decreased the proportion of cells in the S phase by 6.7% and the G2/M phase by 26.6% compared to the control. Treatment with 3.4 µM FA also decreased the proportion of cells in the S phase and G2/M phase compared to the control by 4.9 and 9.4%, respectively. This is consistent with Caco2 cells approaching confluency, according to Ding et al. (1998).  Comparing the data from 3 day and 5 day folate treatment showed a large difference in cell cycle analysis. The proportion of cells in the G1 phase is much higher in Caco2 cells treated with either FA or 5MTHF for 5 days (61.9 – 67.3 %; Table 2.3) compared to the cells treated with the same treatments for 3 days (48.1 – 52.5 %; Table 2.2). On day 5, the folate treated groups had percentages of cells in the S phase ranging from 18 – 22.4% (Table 2.3). The percentage of cells in the S phase of the 3 day folate-treated Caco2 cells was 30.8 – 35.7% (Table 2.2). As explained earlier, as cells approach confluency, the proportion of cells in the G1 phase increases while the proportion of cells in the S phase decreases (Ding et al., 1998). Compared with Caco2 cells treated with FA and 5MTHF at all concentrations for 3 days, 5 day treated Caco2 cells appeared to corroborate this observation. However, the 3 and 5 day treatments were separate experiments. They were done at different times and the 5 day treatment was preceded by a 5 day folate depletion period. These differences in folate treatment regimen made any direct comparison between the two experiments unreliable.   A study in normal human fibroblast cells (GMS03349) explored the effect of folate deficiency on the cell cycle and found that by day 7 of culturing cells in folate-deficient media, there is a change in the proportion of cells in the S phase (Katula et al., 2007). This difference may be attributed to the degree of confluency: as mentioned 67  above, the proportion of Caco2 cells in the G1 phase is inversely related to the proportion of cells in the S phase as cells grow more confluent (Ding et al., 1998).  After 3 and 5 days, both FA and 5MTHF at the concentrations tested did not have an effect on pre-confluent Caco2 cell proliferation determined using the BrdU incorporation assay compared to the control. However, cell cycle analysis shows a spike in the proportion of the cells in the pre-G1 phase after 5 days of 3.4 µM folate treatment compared to the control.  Both assays assess cell proliferation using BrdU incorporation; however the BrdU incorporation assay looks at the whole population of cells while cell cycle analysis uses BrdU as a marker to indicate individual cells engaged in DNA synthesis, a hallmark of the S phase. So the BrdU incorporation assay can be swayed by the number of cells: the more cells there are, the more intense the green signal. A more intense signal may indicate a growth promoting effect or if seeding density was different among treatments or if cells were lost during the assay, it could cause a misleading result.  The results of the BrdU incorporation assay and cell cycle analysis together are an indication of cell proliferation. Based on the results, compared to the control folate treatment, regardless the form and concentrations tested, did not have an effect on cell proliferation. Likely, the lack of an effect is due to the treatment duration. In other experiments, treatments are much longer, starting at 20 days and increasing. Perhaps 3 and 5 day periods were too short to have a significant effect on cell proliferation.  2.4.2. Apparently Differential Effect of FA and 5MTHF on Cell Viability and             Proliferation   Caco2 cells treated with FA at all concentrations tested resulted in a higher level of cell proliferation compared to the cells treated with 5MTHF at the same concentrations 68  after 3 days (Figure 2.3). The differential effects of FA on cell proliferation compared to 5MTHF have not been well explored in cell culture or animal studies, let alone in humans. Perhaps this difference can be attributed to high levels (0.101 mM) of methionine in the culture medium. Total plasma methionine in healthy human adults should be less than 10.5 – 11.7 µmol/L to ensure no increased risk for vascular disease (Stampfer et al., 1992), which is much less concentrated than the cell culture medium. Methionine loading in healthy human subjects leads to increased plasma concentrations of SAM and homocysteine, and a decrease in plasma 5MTHF (Loehrer et al., 1996). The authors theorized that the decreased serum 5MTHF concentration is caused by increased turnover of the homocysteine remethylation reaction that requires 5MTHF as a methyl donor (Loehrer et al., 1996). However, in rat liver, when methionine concentrations are high, it leads to increased SAM levels, which inhibits MTHFR (Krebs et al., 1976). The inhibition of MTHFR increases levels of 5,10-methyleneTHF and other C1-THF derivatives, including 10-formylTHF (Krebs et al., 1976). The increased level of 10-formylTHF increases the activity of formylTHF dehydrogenase (catalyzes the converson of 10-formylTHF to THF) and causes a drop in 5MTHF levels (Krebs et al., 1976). In this way, in the presence of high dietary or extracellular methionine, the cell disposes of excess C1 units and decreases 5MTHF concentrations (Krebs et al., 1976).  Akoglu et al. (2004) observed that hyperproliferation caused by high concentrations of homocysteine (2 µmol/L) was reversed by folate supplementation (5MTHF had a more anti-proliferative effect than folic acid) in Caco2 cells. This study also showed that folate treatment after exposure to high levels of homocysteine led to an increased proportion of Caco2 cells in the G1 phase (Akoglu et al., 2004). In addition, 69  SAM is an allosteric inhibitor of MTHFR, which would lower the amount of 5,10-methyleneTHF converted to 5MTHF (Brosnan and Brosnan, 2006).  Perhaps 5MTHF treatment did not have a proliferative effect on Caco2 cells because of the presence of methionine in the medium. According to the manufacturer, RPMI 1640 medium is formulated with 0.101 mM L-methionine (Gibco, Grand Island, NY). Perhaps methionine doesn’t have the same effect on FA treated cells because FA is reduced first to DHF and then to THF, and it can be used in DNA synthesis reactions.   FA must first be reduced to DHF and further to THF to be able to carry out biological functions in cells. THF is then able to participate in the conversion of deoxyuridylate to thymidylate (as 5,10-methyleneTHF) and the synthesis of purines (as 10-formylTHF) before it acts as a methyl donor to homocysteine (Figure 1.2). 5MTHF, on the other hand, must donate a methyl group to homocysteine to produce methionine before it is able to participate in any of the nucleotide synthesis functions. Perhaps this accounts for the differences in BrdU incorporation observed in Caco2 cells after 3 days treatment with FA or 5MTHF (Figure 2.3). FA enters the cycle in a form that is the precursor for any of the functions of folate. If this were the case, it would take a longer period of time for 5MTHF supplementation to have the initial effects on proliferation decrease. This is exactly what was observed in Caco2 cells after 5 days treatment with either FA or 5MTHF (Figures 2.3, 2.4). After 3 days, cell proliferation was significantly higher at all concentrations of FA compared to the same concentrations of 5MTHF. However, after 5 days of FA treatment there was no difference in cell proliferation compared to cells treated with 5MTHF (Figure 2.4). 70  In lymphocytes isolated from healthy females, a higher frequency of apoptosis and lower cell viability was observed in the cells treated with 5MTHF at 12 or 120 nM for 9 days; but not in cells treated with FA at the same concentrations (Wang and Fenech, 2003). The lowest amount of apoptosis was observed in lymphocytes treated with 120 nM FA and the highest apoptosis levels were observed after treatment with 5MTHF (Wang and Fenech, 2003). In this thesis study, treating Caco2 cells with FA at 0.9 µM for 3 days increased cell viability by 15.8 % compared to the cells treated with 5MTHF at the same concentration (Figure 2.1). In HT29 colon cancer cells, exposure to FA at 0.23 µM for 3 weeks exhibited a faster growth and higher metabolic activity (as measured by the concentration of folate metabolites: SAM, SAH and 5MTHF) than cells exposed to lower levels of FA (Pellis et al., 2008). Thus, this observation fits in with folate’s role in DNA synthesis and the observed increases in cell proliferation and viability in Caco2 cells treated with FA in this research.   Pellis et al. (2008) reported much higher levels of apoptosis in HT29 cells treated with 0.23 µM FA for 3 weeks compared to lower concentration treatments (10 ng/mL; 0.023 µM). This same phenomenon was not observed at 3 days with Caco2 cells (Table 2.2). There was no difference in the proportion of cells in the pre-G1 phase (apoptosis) regardless of the treatment (Table 2.2). The previously mentioned studies in lymphocytes (Wang and Fenech, 2003) and HT29 cells (Pellis et al., 2008) had much longer treatment duration (9 days and 3 weeks, respectively); It seems that a 3 day folate treatment is not long enough to bring about a difference in the proportion of cells in pre-G1 phase.   Caco2 cells treated with a higher concentration of FA (3.4 µM) exhibited a 15.9% increase of cells in the S phase compared to cells treated with 5MTHF. FA treatment 71  resulted in increased cell proliferation at all concentrations tested and increased cell viability at 0.9 µM compared to 5MTHF treatment over 3 days.   A study looking at the effects of different folates in Caco2 cells concluded that 5MTHF, along with DHF, are growth-inhibitory compounds in colon cancer cells (Akoglu et al., 2001). They observed a larger decrease in cell proliferation relative to the control after 48 hour treatment compared to 24 hours (Akoglu et al., 2001). The authors reasoned that after 48 hours, FA had been reduced to the active compounds of growth inhibition, 5MTHF and DHF (Akoglu et al., 2001). If their reasoning holds correct then a longer folate treatment should result in less difference in cell proliferation between cells treated with FA and 5MTHF. After 3 days treatment with 3.4 µM FA or 5MTHF, 5MTHF treated cells had decreased proliferation. After 5 days there was no statistical difference among any of the groups.  One of the reasons for using FA as a fortificant is because of its stability: FA can stand long periods of storage, light, and cooking temperatures. 5MTHF is much more susceptible to oxidation than FA. The observed difference in cell proliferation and cell viability could be attributed to the stability of 5MTHF in the treatment medium. 5MTHF is oxidized first to 5-methyldihydrofolate (5MDHF) and then to p-aminobenzoylglutamate (the result of C9-N10 scission), after which reducing agents cannot rescue it back to its original form (Ng et al., 2008). 5MTHF is more stable at acidic pH than at neutral or basic pH (Ng et al., 2008) and its thermal stability is higher as well (Liu et al., 2011). Addition of ascorbic acid to 5MTHF-containing solutions increases the salvage of 5MTHF and reduces oxidative break down, however after 426 minutes in pH 7 buffer, 50% of 5MTHF is lost, a significant amount (Ng et al., 2008). 72  Sodium ascorbate can regenerate 5MTHF after is has been exposed to heat (Liu et al., 2011. Addition of sodium ascorbate can regenerate 5MTHF to 93 ± 3% and 87 ± 4% after heating at 50°C for 60 and 150 minutes, respectively (Liu et al., 2011). A study measuring serum folate response to oral doses of 5MTHF in healthy adult men found that administering 5MTHF with L-ascorbic acid results in significantly higher area under the curve folate response compared to administering 5MTHF alone (Verlinde et al., 2008). When treating Caco2 cells, the utmost care was taken to ensure as little damage to 5MTHF as possible. Stock solutions were made containing 5% sodium ascorbate, stock solutions were aliquotted and frozen immediately (aliquotted stock was used within 1 month) and stock solutions were protected from the light. Despite precautions taken, some 5MTHF was likely oxidized in the treatment medium, meaning less 5MTHF was available for Caco2 cells in the experiments than FA, which is stable in the medium.  This could have resulted in reduced viability and proliferation in the 5MTHF treated Caco2 cells.  Difference in the bioavailability of 5MTHF and FA likely cannot explain differences in observed cell proliferation, as 5MTHF is equal to or more bioavailable than FA. As discussed in Chapter 1, natural folate has a polyglutamate tail which must be hydrolyzed before the folate can be absorbed. As a result naturally occurring dietary folates (with polyglutamate tails) have a lower bioavailability than FA. The 5MTHF used in this thesis research is a synthetic, monoglutamylated form. Studies comparing the bioavailability of 5MTHF and FA in humans have shown that 5MTHF has equivalent or higher bioavailability compared to FA.  In women of childbearing age there was no difference in plasma folate or red blood cell (RBC) folate after supplementation with FA 73  and 5MTHF (Venn et al., 2002). In a double-blind, crossover study, 13 healthy men who were presaturated with FA received placebo capsules, 500 µg FA capsules, or 500 µg 5MTHF capsules for a 1 week interval in random order (Pentieva et al., 2004). There was no difference in maximum plasma folate response or area under the curve for plasma folate between the two folate treatments (Pentieva et al., 2004). Lamers et al. (2006) conducted a double-blind, randomized, placebo-controlled intervention study in which healthy women aged 19-33 years received 400 µg FA, 416 µg 5MTHF, 208 µg 5MTHF, or placebo daily for 24 weeks. RBC folate increased significantly more with 416 µg 5MTHF supplementation compared to 400 µg FA and 208 µg 5MTHF (Lamers et al., 2006). It was also found that the short term bioavailability of 5MTHF was higher compared to FA in healthy men and women (in a repeated measures crossover design experiment) as measured by plasma folate (Harvey, 2011). In a randomized, crossover study, healthy women received a single oral dose of 400 µg FA and 416 µg 5MTHF (Prinz-Langenohl et al., 2009). Prinz-Langenohl et al. (2009) found that area under the curve of plasma folate and maximum plasma folate concentration were both significantly higher for 5MTHF compared to FA. Studies involving 5MTHF and FA in humans suggest equal or better bioavailability of 5MTHF compared to FA. However, in Caco2 cells, no folate uptake studies were performed, so it cannot be ruled out that folate uptake differences played a role in the differences observed in cell proliferation after 3 days treatment with FA and 5MTHF.     74  2.4.3 The Effect of Folate Treatment on the Abundance of Folate Transporters  In this study, folate form and concentration had no significant effect on the abundance of PCFT, RFC (Figure 2.5), FRα (Figure 2.6) and BCRP (Figure 2.7). These observations differ from previous studies that have shown that these transporters are affected by extracellular folate concentration. The presence of PCFT protein in pre-confluent Caco2 cells was not detectable in this study. A possible cause was a low abundance of PCFT in pre-confluent Caco2 cells.  Subramanian et al. (2008) reported low levels of PCFT in pre-confluent Caco2 cells, which possess a colonocyte phenotype. Upon confluence PCFT expression increases as the cells differentiate into a phenotype resembling that of a small intestinal enterocyte (Subramanian et al., 2008). Since this study used undifferentiated Caco2 cells that don’t possess a small intestinal enterocyte phenotype, PCFT may not have been expressed in high enough levels to be detected. The lack of signal for PCFT could also be due to the assay not working. This remained as a possibility as there was no positive control used during the assay.   It was observed that Pcft mRNA in laying hens was downregulated in jejunal tissue when the chickens were fed a diet supplemented with 5MTHF compared to chickens fed the control diet, whereas chickens fed a FA-supplemented diet showed no change compared to chicken fed the control diet (Jing et al., 2010). Other groups have shown the folate status affects the expression of PCFT, for example in Caco2 cells supplemented with 0.25, 9 and 100 µM FA, PCFT mRNA expression decreases with rising folate concentration (Ashokkumar et al., 2007). However, both studies only 75  reported PCFT mRNA expression, which does not necessarily correlate with protein abundance. RFC abundance was not affected by folate concentration or form (Figure 2.5). These observations are inconsistent with what has been previously observed. For example, Caco2 cells treated with 0.25, 9 and 100 µM FA for 5 passages showed an inverse relationship between FA concentration and RFC protein and mRNA abundance (Ashokkumar et al., 2007). The relative abundance of protein between cells treated with 0.25 µM FA (140%) and 100 µM FA (50%) is approximately 3:1 (Ashokkumar et al., 2007). This ratio is similar to what was observed in Caco2 cells after 5 days treatment with 3.4 µM 5MTHF compared to the control (Figure 2.5). The Caco2 cells in the Ashokkumar et al. (2007) study were harvested after 5 generations of treatment and 3-4 days post-confluence. At this point the Caco2 cells have begun to differentiate. Other groups have shown that RFC mRNA is upregulated in Caco2 cells exposed to 0 folate conditions (Cockman, 2009; Subramanian et al., 2003) and also in human colonic epithelial cell lines (Crott et al., 2008).  As RFC is upregulated in folate deficiency, one would expect that RFC would be most abundantly expressed after 10 days culture in very low folate conditions (control group). The expression of RFC in Caco2 cells changes depending on the confluency of the cells (Subramanian et al., 2008). In Caco2 cells, confluent and post-confluent cells have much higher relative mRNA expression of RFC compared to preconfluent cells, and this was confirmed by Western blot (Subramanian et al., 2008). No difference was observed in RFC relative protein abundance between the treated groups and the control. Based on the evidence in the literature, a difference in RFC protein levels between folate 76  concentrations was expected. Given that the cells were harvested before confluency and thus before differentiation took place, this could explain the lack of difference in RFC protein levels among different treatments. The degree of differentiation is important as the cells take on a different phenotype, transforming from a cancerous colonocyte to a cell with a small intestinal cell phenotype. Folate is absorbed largely in the small intestine, where the folate transporters are found in higher abundance than in the large intestine. FRα abundance was not significantly affected by folate treatment in Caco2 cells in this study (Figure 2.6). FRα has been previously shown to be upregulated in folate deficient Caco2 cells (Cockman, 2009), in folate deficient renal HK-2 cells (Ashokkumar et al., 2007) and human colonic epithelial cell lines treated with 25 nM FA compared to 50, 75 and 150 nM FA (Crott et al., 2008). With this in mind, it was expected that the control group would have the highest relative abundance of FRα, however no differences were observed among any of the groups.  BCRP expression is not affected by Caco2 confluency and it is found on the apical membrane (Xia et al., 2005). After 5 days culture in free folate medium followed by folate treatment for 5 days, expression of BCRP was not affected by folate form or concentration (Figure 2.7). BCRP has been found to be induced in folate deficient Caco2 cells (Lemos et al., 2008). It has also found to be either up-regulated or down-regulated by methylation of the promoter region of the BCRP gene in lung cancer cells (Nakano et al., 2008), renal carcinoma (To et al., 2006), pancreatic cancer cell lines (Chen et al., 2012) and multiple myeloma cell lines and patient plasma cells (Turner et al., 2006). The evidence from other cancer cell lines points to a possibility that BCRP abundance 77  depends on the methylation status of CpG islands in the promoter region. If this is the case in colon cancer, it could explain the upregulation in folate deficiency. However, in this study, the control Caco2 cells did not show increased BCRP expression relative to folate supplemented cells (Figure 2.7). If BCRP is indeed regulated by promoter methylation, the control cells would be expected to have the highest abundance of BCRP, especially considering that BCRP expression is apparently not affected by Caco2 cell confluency. However, BCRP is regulated by promoter methylation in different tissues, for example, lung cancer, renal carcinoma, and pancreatic cancer. Promoter methylation is tissue specific, so what is true for lung cancer cells, for example, is not necessarily true for colorectal adenocarcinoma cells. The abundance of folate transporters was unaffected by folate form or concentration in Caco2 cells after 5 days of folate treatment. Perhaps 5 days of folate treatment was not enough time to elicit a change in the abundance of folate transporters. In other experiments, folate treatment has been considerably longer than 5 days. For example, Ashokkumar et al. (2007) found a difference in the expression of RFC, PCFT and FRα after maintaining the cells in folic acid for 5 generations in Caco2 and HK-2 cells. Crott et al. (2008) found differences in the expression of RFC (SLC19A1) and FRα (FOLR1) after treating cells for 32-34 days.  The response of Caco2 cells to folate treatment was surprising as it did not correlate with what was previously been found in the literature for each transporter. Perhaps there is something else at work. Due to the instability of 5MTHF, it is likely that some was oxidized in the medium. Perhaps the oxidative breakdown products of 5MTHF have an effect on the abundance of folate transporters. Unfortunately, there seems to be 78  no published studies about the effect of 5-methylDHF and p-aminobenzoylglutamate on folate transporter abundance at this time.  2.4.4 Nuclear Localization of β-Catenin was Unaffected by Folate Treatment   β-Catenin nuclear localization can be used as a measure of Wnt signalling pathway activity because translocation of this protein into the nucleus is an essential step in the pathway (Archbold et al., 2011). Once inside the nucleus, β-Catenin complexes with transcription factors TCF/LEF1 to regulate gene transcription (Archbold et al., 2011).  Studies have shown that the localization of β-Catenin can be affected by folate treatment. Morillon II (2008) showed that β-Catenin nuclear localization increased in NIH3T3 cells treated with 0 folate for 10 days compared to cells treated with a “sufficient” [sic] amount of folate. Another study found that FA supplementation (5 mg daily for 1 year) in patients with adenomatous polyps decreased β-Catenin nuclear localization in primary colorectal tissue when compared to baseline measurements (Jaszewski et al., 2004).  However, in this research, folate treatment had no significant effect on β-Catenin nuclear localization (Figures 2.8, 2.9). This absence of decreased β-Catenin nuclear localization in response to folate treatment could be due to a combination of a relatively short duration of folate treatment and low folate concentration.  Furthermore, the responsiveness of cells to folate status-related β-Catenin cellular translocation may also be cell type specific. To date, there have been no studies investigating the effect of different forms of folate on β-Catenin nuclear localization. This 79  should be investigated more thoroughly involving longer duration of folate treatment and higher folate concentration.   2.4.5 Summary  In summary, this thesis project showed that, after 3 days of folate treatment, Caco2 cells treated with FA had increased cell proliferation at all three concentrations tested and increased cell viability at 0.9 µM compared to the cells treated with 5MTHF at the same concentration. After 5 days of folate depletion followed by 5 days folate treatment, there was no difference of cell viability or proliferation among Caco2 cells treated with FA or 5MTHF at any concentration. The mechanism behind the increases in cell proliferation and cell viability after treatment with FA compared to 5MTHF is unclear. The relative abundance of RFC, FRα and BCRP was not affected by the form of folate at the concentrations tested, which was contrary to what has been previously observed in the literature. β-Catenin nuclear localization was also not affected by the form of folate at the concentrations tested. However, many studies used longer folate treatment duration at higher folate concentrations; perhaps these differences in the folate treatment regimen played a role in the incongruous results.  Reflecting back, treatment duration was too short and a chronic folate treatment model should have been used instead.      80  Table 2.1: Antibodies used in the Western blots.  Target Protein Antibody1 Dilution Catalogue Number BCRP 1°: Rabbit anti-ABCG2 polyclonal IgG  1:1,000 sc-25821 2°: Bovine anti-rabbit IgG-HRP 1:5,000 sc-2370 RFC 1°: Rabbit anti-RFC1 polyclonal IgG 1:400 sc-98971 2°: Bovine anti-rabbit IgG-HRP 1:2,500 sc-2370 FRα 1°: Rabbit anti-FR polyclonal IgG 1:300 sc-28997 2°: Bovine anti-rabbit IgG-HRP 1:5,000 sc-2370 PCFT 1°: Goat anti-HCP1 polyclonal IgG 1:300 sc-54204 2°: Donkey anti-goat IgG-HRP 1:2,500 sc-2020 α-Tubulin  (loading control) 1°: Mouse anti-α tubulin monoclonal IgG2A 1:300 sc-5286 2°: Goat anti-mouse IgG2A-HRP 1:1,000 sc-2061 1 All antibodies were obtained from Santa Cruz Biotechnology.                              81  Table 2.2: Cell cycle analysis of Caco2 cells following 3 day treatment with FA or 5MTHF.  Sample Pre-G1 phase G1 Phase S phase G2/M Phase Control 5.7 52.5 34.1 8.6 0.9 µM FA 6.0 (+5.3 %) 49.9 (-5 %) 35.4 (+3.8 %) 9.3 (+8.1 %) 2.3 µM FA 6.5 (+14 %) 51.1 (-2.7 %) 33.6 (-1.5 %) 9.1 (+5.8 %) 3.4 µM FA 6.2 (+8.8 %) 48.1 (-8.4 %) 35.7 (+4.7 %) 12 (+39.5 %) 0.9 µM 5M 7.7 (+35.1 %) 52.2 (0 %) 33.5 (-1.8 %) 10.6 (+23.2 %) 2.3 µM 5M 5.1 (-10.5 %) 49.9 (-5 %) 32.9 (-3.5 %) 11.9 (+38.4 %) 3.4 µM 5M 6.2 (+8.8 %) 52.2 (0 %) 30.8 (-9.7 %) 12.4 (+44.2 %) Data from cell cycle analysis of Caco2 cells after 3 days treatment with FA or 5MTHF. Caco2 cells were cultured in folate-free RPMI-1640 medium containing 0, 0.9, 2.3, or 3.4 µM FA or MTHF for 3 days. Cell cycle was determined using BrdU incorporation for S phase detection and PI staining for total DNA and analyzed by flow cytometry. The table shows proportion of cells in each phase. Change relative to the control is in brackets. Scatter plots and histograms for cell cycle analysis are shown in Figure A.1. Abbreviations: FA (folic acid), 5M (5-methyltetrahydrofolate).                        82  Table 2.3: Cell cycle analysis of Caco2 cells following 5 day treatment with FA or 5MTHF. Sample Pre-G1 phase G1 phase S phase G2/M phase Control 3.8 62.3 22.4 12.8  0.9 µM FA 3.7 (-2.6 %) 67.3 (+8 %) 18 (-19.6 %) 11.8 (-7.8 %) 2.3 µM FA 2.8 (-26.3 %) 61.9 (-0.6 %) 21.9 (-2.2 %) 12.5 (-2.3 %) 3.4 µM FA 13.6 (+ 3.6 fold) 63.4 (+1.8 %) 21.3 (-4.9 %) 11.6 (-9.4 %) 0.9 µM 5M 3.8 (0 %) 67.3 (+8 %) 18.5 (-17.4 %) 11.9 (-7 %) 2.3 µM 5M 3.5 (-7.9 %) 63.6 (+2.1 %) 20.6 (-8 %) 13.5 (+5.5 %) 3.4 µM 5M 20.3 (+ 5.3 fold) 64 (+2.7 %) 20.9 (-6.7 %) 9.4 (-26.6 %) Data from cell cycle analysis of Caco2 cells after 5 days treatment with FA or 5MTHF. Caco2 cells were cultured in folate-free RPMI-1640 medium for 5 days and then transferred to folate-free RPMI-1640 medium containing 0, 0.9, 2.3, or 3.4 µM FA or 5MTHF for 5 days. Cell cycle was determined using BrdU incorporation for S phase detection and PI staining for total DNA and analyzed by flow cytometry. The table shows proportion of cells in each phase. Change relative to the control is in brackets. Scatter plots and histograms for cell cycle analysis are shown in Figure A.2. Abbreviations: FA (folic acid), 5M (5-methyltetrahydrofolate).              83    Figure 2.1: Cell viability of Caco2 cells after 3 day treatment with FA or 5MTHF. Caco2 cells were cultured in folate-free RPMI-1640 medium containing 0, 0.9, 2.3, or 3.4 µM FA or 5MTHF for 3 days. Cell viability was quantified using the WST-1 colorimetric assay where absorbance is equivalent to cell viability. The values represent mean ± SEM (n=10). Means marked with * are significantly different from the corresponding concentration of 5MTHF (p<0.05). Abbreviations: FA (folic acid), 5M (5-methyltetrahydrofolate).     00.20.40.60.811.21.41.6Cell Viability (O.D. 450) Folate Treatment Control0.9 µM FA2.3 µM FA3.4 µM FA0.9 µM 5M2.3 µM 5M3.4 µM 5M* 84    Figure 2.2: Cell viability of Caco2 cells after 5 day treatment with FA or 5MTHF. Caco2 cells were cultured in folate-free RPMI-1640 medium for 5 days and then transferred to folate-free RPMI-1640 medium containing 0, 0.9, 2.3, or 3.4 µM FA or 5MTHF for 5 days. Cell viability was quantified using the WST-1 colorimetric assay where absorbance is equivalent to cell viability. The values represent mean ± SEM (n=10). No significant differences were observed among the treatments (p<0.05). Abbreviations: FA (folic acid), 5M (5-methyltetrahydrofolate).     00.511.522.533.5Folate TreatmentCell Viability (O.D. 450) Control0.9 µM FA2.3 µM FA3.4 µM FA0.9 µM 5M2.3 µM 5M3.4 µM 5M85    Figure 2.3: Cell proliferation of Caco2 cells after 3 day treatment with FA or 5MTHF. Caco2 cells were cultured in folate-free RPMI-1640 medium containing 0, 0.9, 2.3, or 3.4 µM FA or 5MTHF for 3 days. Cell proliferation was quantified using the BrdU incorporation assay where absorbance is a measure of BrdU incorporation. The values represent mean ± SEM (n=10). Means marked with capital letters are significantly different from the control, while means marked with * are significantly different from the corresponding concentration of 5MTHF (p<0.05). Abbreviations: FA (folic acid), 5M (5-methyltetrahydrofolate).      00.10.20.30.40.50.60.70.8Folate TreatmentCell Proliferation (O.D. 405) Control0.9 µM FA2.3 µM FA3.4 µM FA0.9 µM 5M2.3 µM 5M3.4 µM 5MA B * * * 86   Figure 2.4: Cell proliferation of Caco2 cells after 5 day treatment with FA or 5MTHF. Caco2 cells were cultured in folate-free RPMI-1640 medium for 5 days and then transferred to folate-free RPMI-1640 medium containing 0, 0.9, 2.3, or 3.4 µM FA or 5MTHF for 5 days. Cell proliferation was quantified using the BrdU incorporation assay where absorbance is a measure of BrdU incorporation. The values represent mean ± SEM (n=10). No significant differences were observed among the treatments (p<0.05). Abbreviations: FA (folic acid), 5M (5-methyltetrahydrofolate).                       00.10.20.30.40.50.60.70.80.9Folate TreatmentCell Proliferation (A405 - A490) Control0.9 µM FA2.3 µM FA3.4 µM FA0.9 µM 5M2.3 µM 5M3.4 µM 5M87  A)  RFC                                         Control         0.9 µM FA    2.3 µM FA   3.4 µM FA   0.9 µM 5M    2.3 µM 5M     3.4 µM 5M α-tubulin    B)     Figure 2.5: Relative protein abundance of RFC in Caco2 cells after 5 day treatment with FA or MTHF. Caco2 cells were cultured in folate-free RPMI-1640 medium for 5 days and then transferred to folate-free RPMI-1640 medium containing 0, 0.9, 2.3, or 3.4 µM FA or 5MTHF for 5 days. RFC in whole cell lysate was determined using Western blot. Values represent the mean ± SEM (n=3). Western blot results (A) were analyzed for the ratio of RFC/α-tubulin (B). No significant difference among the treatment groups was observed (p<0.05) Abbreviations: FA (folic acid), 5M (5-methyltetrahydrofolate).            00.511.522.533.544.5Control 0.9 µM FA 2.3 µM FA 3.4 µM FA 0.9 µM 5M 2.3 µM 5M 3.4 µM 5MNormalized Density (Fold of control) 88  A)  FRα                                Control              0.9 µM FA          2.3 µM FA      3.4 µM FA        0.9 µM 5M       2.3 µM 5M   3.4 µM 5M α-tubulin   B)  Figure 2.6: Relative protein abundance of FRα in Caco2 cells after 5 day treatment with FA or MTHF. Caco2 cells were cultured in folate-free RPMI-1640 medium for 5 days and then transferred to folate-free RPMI-1640 medium containing 0, 0.9, 2.3, or 3.4 µM FA or 5MTHF for 5 days. FRα in whole cell lysate was determined using Western blot. Values represent the mean ± SEM (n=3). Western blot results (A) were analyzed for the ratio of FRα/α-tubulin (B). No significant difference among the treatment groups was observed (p<0.05) Abbreviations: FA (folic acid), 5M (5-methyltetrahydrofolate).        0.01.02.03.04.05.06.07.08.0Control 0.9 µM FA 2.3 µM FA 3.4 µM FA 0.9 µM 5M 2.3 µM 5M 3.4 µM 5MNormalized Density (Fold of control) 89  A)  BCRP  Control        0.9 µM FA     2.3 µM FA     3.4 µM FA   0.9 µM 5M  2.3 µM 5M   3.4 µM 5M α-tubulin   B)   Figure 2.7: Relative protein abundance of BCRP in Caco2 cells after 5 day treatment with FA or MTHF. Caco2 cells were cultured in folate-free RPMI-1640 medium for 5 days and then transferred to folate-free RPMI-1640 medium containing 0, 0.9, 2.3, or 3.4 µM FA or 5MTHF for 5 days. BCRP in whole cell lysate was determined using Western blot. Values represent the mean ± SEM (n=4). Western blot results (A) were analyzed for the ratio of BCRP/α-tubulin (B). No significant difference among the treatment groups was observed (p<0.05) Abbreviations: FA (folic acid), 5M (5-methyltetrahydrofolate).         00.20.40.60.811.21.41.61.8Control 0.9 µM FA 2.3 µM FA 3.4 µM FA 0.9 µM 5M 2.3 µM 5M 3.4 µM 5MNormalized Denisty (Fold of control) 90   Figure 2.8: β-Catenin fluorescence (ratio of nucleus to cytoplasm) in Caco2 cells after 5 days treatment with FA or 5MTHF. Caco2 cells were cultured in folate-free RPMI-1640 medium for 5 days and then transferred to folate-free RPMI-1640 medium containing 0, 0.9, 2.3, or 3.4 µM FA or 5MTHF for 5 days. β-Catenin was visualized with Alexa Fluor 488® and nuclei were counterstained with DAPI. β-Catenin fluorescence is represented by the ratio of nuclear to cytoplasmic fluorescent and normalized to the control. All values are mean ± SEM (n=5 except: n=3 for 0.9 µM 5M, n=4 for 3.4 µM FA and 5M). No significant difference among the treatment groups was observed (p<0.05). Abbreviations: FA (folic acid), 5M (5-methyltetrahydrofolate).           00.20.40.60.811.21.41.6β-Catenin Fluorescence Ratio of Nucleus to Cytoplasm (fold of control) Folate Treatment Control0.9 µM FA2.3 µM FA3.4 µM FA0.9 µM 5M2.3 µM 5M3.4 µM 5M91          A      B     C                                                             Control 0.9 µM FA 2.3 µM FA 3.4 µM FA 92                                               Figure 2.9: β-Catenin fluorescence in Caco2 cells after 5 days treatment with FA or 5MTHF. Caco2 cells were cultured in folate-free RPMI-1640 medium for 5 days and then transferred to folate-free RPMI-1640 medium containing 0, 0.9, 2.3, or 3.4 µM FA or 5MTHF for 5 days. β-Catenin was visualized with Alexa Fluor 488® (green) and nuclei were counterstained with DAPI (blue). Images captured using confocal microscopy. (A) Alexa Fluor 488® and DAPI; (B) DAPI only; (C) Alexa Fluor 488® only. Abbreviations: FA (folic acid), 5M (5-methyltetrahydrofolate).      0.9 µM 5M 2.3 µM 5M 3.4 µM 5M 93  CHAPTER 3 Limitations and Future Directions  3.1 Limitations  There are several limitations of this research project. One of the limitations was the use of an in vitro system. Although a single-cell system is useful in identifying cellular and molecular targets in the early phase of research, it eliminates the cell-cell communication, interactions, and coordination, and regulation as seen in a multicellular organism system. It is advantageous that the test conditions are relatively easier to control in an in vitro system and thus specific phenomena can be examined in more detail, but it is a major limitation because results obtained from an in vitro system cannot be extrapolated to whole organisms or even other cell types or cell lines of the same tissue. So results obtained from studies in in vitro systems need to be confirmed in in vivo afterwards. This research needs to be repeated in other colon cancer cell lines as well as in primary culture and hopefully in the future, an in vivo model such as rats or mice.   Another limitation of using an in vitro system is that the cell lines have often been transformed to make them immortal. Transformed cells may not have the same characteristics as the original cells in vivo. They may have altered biochemical and cellular characteristics than their in vivo counterparts. Therefore, results obtained from studies with in vitro system are to be treated with caution.  A third limitation is the stability of 5MTHF. 5MTHF is very labile to light, heat and oxidation. Though precautions were taken to prevent oxidation of the compound, it is impossible to fully prevent it from occurring. The oxidation of 5MTHF may have resulted in less 5MTHF in the culture medium and it could have a direct influence on the 94  results obtained. 5MTHF is first oxidized to 5MDHF, a reversible reaction, and then further oxidized to p-aminobenzoylglutamate, which cannot be rescued by the presence of reducing agents (Ng et al., 2008). These oxidation products are not functional as folates. The conditions within the lumen of the small intestine (where folate is absorbed) and the large intestine are much different than what the Caco2 cells used in this research are exposed to. These conditions would affect the folate treatments differently. For example, intestinal cells can produce a reducing environment which would affect the stability of 5MTHF. These conditions were not recreated in this study. Caco2 cells were used in my thesis research not for their differentiation potential, but because they are colorectal adenocarcinoma cells. In vivo, colorectal adenocarcinoma cells would be subjected to the conditions within the lumen of the large intestine, as opposed to the conditions in the experiments: 37°C humidified incubator, 5% CO2 atmosphere, and cell medium. Therefore, the conditions of the experiments do no replicate the in vivo conditions that an actual colorectal adenocarcinoma would be exposed to. Another limitation related to folate treatment is that high folate concentrations (0.9 – 3.4 µM) were used. These concentrations were chosen to represent concentrations that the cells may be exposed to on the apical membrane, in other words, supplemental folate. However, systemic folate levels are much lower than the concentrations of folate used in this research, for example, the mean serum folate in healthy young women (18 – 25 years) was 33.1 nmol/L (Shuaibi et al., 2008). So serum folate levels are much lower than the treatment concentrations used in this research. 95   Many studies performed with colon cancer cell lines have longer treatment and culture times. For this project, treatment time was capped at 5 days. Caco2 cells have a relatively slow doubling time of 62 hours, according to the ATCC® product specifications for Caco2 (ATCC® HTB-37™). Perhaps treating the cells for a longer time could reduce variability and bring about more significant results.  Other studies examining the link between folate and cancer cell growth have found an effect of folate after longer treatment periods. Therefore, longer treatment durations should be investigated. For example, a study in HT29 cells showed that treatment with 0.23 µM FA for 3 weeks had growth and metabolism promoting effect compared to treatment with 0.023 µM FA (Pellis et al., 2008). Another study in Caco2 cells found an inverse relationship with FA concentration in the medium and RFC mRNA and protein abundance after 5 passages of Caco2 cells (Ashokkumar et al., 2007). The growth rate of Caco2 cells was decreased when grown in folate deficient [sic] conditions compared to folate sufficient [sic] conditions after 15 and 30 days (Cockman, 2009). This same study also found differences in RFC mRNA abundance, β-Catenin transcripts, and β-Catenin nuclear localization in Caco2 cells in folate deficient or sufficient conditions after 30 days (Cockman, 2009).  3.2 Future Directions The main hypothesis for the interactions between folate, Wnt signalling and colon cancer is thought to be related to the regulation of genes. Future directions for this research should include increased treatment length, different types of cells, studying the 96  expression of key genes in folate transport and metabolism, the Wnt signalling pathway as well as colorectal cancer progression.   3.2.1 Treatment Length As rationalized above, the treatment duration for the current study was probably too short. Results of other studies have shown an effect of folate with longer treatment durations. Therefore, the present study should be repeated using longer treatment times. Based on the results of other studies, a range of time points would be ideal, for example, 0, 7, 14, 21, 28, and 35 days. However, if using Caco2 cells it must be ensured that the cells are not allowed to differentiate or else they will lose their adenocarcinoma phenotype. After looking at that range, depending on the results, a narrower span of treatment times can be chosen.  3.2.2 Cell Type  In this thesis research, only one cell type was used. In future studies, other cell lines should be used. ATCC® offers a spectrum of colon cancer cell lines, grouped together specifically because of genomic mutations affecting specific genes. For example, Colon Cancer Panel 2, BRAF (ATCC® TCP-1007™) is a panel of 8 colon cancer cell lines with mutations in one or more of BRAF, APC, CTNNB1, EGFR, FBXW7, NF1, PIK3CA, PIK3R1, SMAD4, and TP53. APC and CTNNB1 are genes that are essential in the Wnt signalling pathway. This panel contains SNU-C1 (ATCC® CRL-5972™), SW48 (ATCC® CCL-231™), RKO (ATCC® CRL-2577™), COLO 205 (ATCC® CCL-222™), SW1417 (ATCC® CCL-238™), LS411N (ATCC® CRL-2159™), NCI-H508 97  (ATCC® CCL-253™), and HT-29 (ATCC® HTB-38™). Testing other cell lines is important because what occurs in one cell type may not in another. Each cell line has its own unique features, for example SW48 is classified as a Duke`s type C, grade IV colorectal adenocarcinoma with an epithelial cell morphology, isolated from an 82-year old Caucasian female (Product Specifications; ATCC® CCL-231™). The present study only examined the effects of folate type in Caco2 cells. Researching one cell type gives limited data that can’t be readily applied to other cell lines or cell types.   It would be interesting to contrast findings from the cancerous cell lines with healthy cells. There are 5 “normal” cell lines available from ATCC®: CCD 841 CoN (ATCC® CRL-1790™), FHC (ATCC® CRL-1831™), CCD-19Co (ATCC® CRL-1459™), CCD-33Co (ATCC® CRL-1539™), and CCD-112CoN (ATCC® CRL-1541™). These cell lines are all isolated from very young patients, the oldest being 7 years (CCD-112CoN) and the youngest a 13 week gestation fetus (FHC), according to the product specifications (ATCC®).   3.2.3 Gene Expression   One of the intended endpoints of this thesis research was gene expression of the folate transporters and of Wnt signalling pathway components in response to different folate treatments. Because other studies have showed that extracellular folate concentrations affect the expression of folate transport genes, future research projects should examine whether there is a difference in expression of folate transport genes after treatment with different forms of folate. In addition, to further study the involvement of Wnt and folate in colon cancer, the original plan in this thesis research was to examine 98  the effect of folate type and concentration on expression of genes within the Wnt signalling pathway using a human Wnt signalling pathway PCR array (PAHS-043A; SA Biosciences, Frederick, MD).                                         99  References  "8 Folate ." Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline . Washington, DC: The National Academies Press, 1998 Aguilera O, Fraga MF, Ballestar E et al. Epigenetic inactivation of the Wnt antagonist DICKKOPF-1 (DKK-1) gene in human colorectal cancer. Oncogene 2006;25:4116-21.  Akoglu B, Faust D, Milovic V et al. Folate and chemoprevention of colorectal cancer: Is 5-methyltetrathydrofolate an active antiproliferative agent in folate-treated colon-cancer cells? Nutrition 2001;17:652-653.  Akoglu B, Milovic V, Caspary WF et al. Hyperproliferation of homocysteine-treated colon cancer cells is reversed by folate and 5-methyltetrahydrofolate. Eur J Nutr 2004;93-99.  Anderson RGW, Kamen BA, Rothberg KG, et al. Potocytosis: sequestration and transport of small molecules by caveolae. Science 1992;255:410-411.  Antony AC. Folate Receptors. Annu Rev Nutr 1996;16:501-521.  Archbold HC, Yang YX, Chen L et al. How do they do Wnt they do?: Regulation of transcription by the Wnt/β-catenin pathway. Acta Physiol  2012;204:74-109.  Ashokkumar B, Mohammed ZM, Vaziri ND, et al. Effect of folate oversupplementation on folate uptake by human intestinal and renal epithelial cells. Am J Clin Nutr 2007;86:159-166.  Austin RC, Lentz SR, Werstuck GH. Role of hyperhomocysteinemia in endothelial dysfunction and atherothrombotic disease. Cell Death and Differentiation 2004;11:S56-S64.  Bafico A, Liu G, Yaniv A et al. Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow. Nature Cell Biol 2001;3:683-86.  Balamurugan K, Said HM. Role of reduced folate carrier in intestinal folate uptake. Am J Physiol Cell Physiol 2006;291:189-193.  Bänzinger C, Soldini D, Schütt C et al. Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 2006;125:509-22.  Baron R, Rawadi G, Roman-Roman S. Wnt signaling: A key regulator of bone mass. Curr Topics in Dev Biol 2006;76:103-27.  Bartscherer K, Pelte N, Ingelfinger D et al. Secretion of Wnt ligands requires Evi, a conserved transmembrane protein. Cell 2006;125:523-33. 100   Bauldauff RL. The relationship between unmetabolized folic acid and serum folate concentrations and cancer risk in older US adults. M.Sc. Thesis 2013. Byrdine F. Lewis  School of Nursing and Health Sciences, Georgia State University.  Bernabei PA, Bensinger WI. Effect of (dl)-5-methyltetrahydrofolate on lymphoid leukemia cell lines. Leukemia Researchi 1991;15:645-49.  Berry RJ, Li Z, Erickson JE, et al. Prevention of neural-tube defects with folic acid in china. N Engl J Med 1999;341:1485-1490.  Bienz M, Clevers H. Linking colorectal cancer to Wnt signaling. Cell 2000;103:311-20.  Blom HJ. Folic acid, methylation and neural tube closure in humans. Birth Defects Research (Part A) 2009;85:295-302.  Blount BC, Mack MM, Wehr CM et al. Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: Implication for cancer and neuronal damage. Proc Nat’l Acad Sci, USA 1997;94:3290-3295.  Bodmer WF, Bailey CJ, Bussey HJR et al. Localization of the gene for familial adenomatous polyposis on chromosome 5. Nature 1987;328:614-6.  Boon EMJ, van der Neut R, van de Wetering M et al. Wnt signaling regulates expression of the receptory tyrosine kinase Met in colorectal cancer. Cancer Res 2002;62:5126-28.  Brabletz T, Jung A, Dag S et al. β-catenin regulates the expression of the matrix metalloproteinase-7 in human colorectal cancer. Am J Path 1999;155:1033-38.  Brosnan JT, Brosnan ME. The sulfur-containing amino acids: An overview. J Nutr 2006;136:1636S-40S.  Brouwer IA, van Dusseldorp M, West CE et al. Bioavailability and bioefficacy of folate and folic acid in man. Nutrition Research Reviews 2001;14:267-93.  Brouwer IA, van Dusseldorp M, Thomas CMG, et al. Low-dose folic acid supplementation decreases plasma homocysteine concentrations: a randomized trial. Am J Clin Nutr 1999;69:99-104.  Carter M, Chen X, Slowinska B et al. Crooked tail (Cd) model of human folate-responsive neural tube defects is mutated in Wnt coreceptor lipoprotein receptor-related protein 6. Proc Natl Acad Scie USA 2005;102:12843-48.  Cedar H. DNA methylation and gene activity. Cell 1988;53:3-4.  101  Chambers JC, Obeid OA, Kooner JS. Physiological increments in plasma homocysteine induce vascular endothelial dysfunction in normal human subjects. Arterioscler Thromb Vasc Biol 1999;19:2922-27.  Chen M, Xue X, Wang F et al. Expression and promoter methylation analysis of ATP-binding cassette genes in pancreatic cancer. Oncology Reports 2012;27:265-69.  Chen Z, Robey RW, Belinsky MG, et al. Transport of methotrexate, methotrexate polyglutamates, and 17β-estradiol at R482 on methotrexate transport. Cancer Res 2003;63:4048-4054.  Chien AJ, Conrad WH, Moon RT. A wnt survival guide: From flies to human disease. J Invest Dermatol 2009;129:1614-27.  Choi SC, Han JK. Xenopus Cdc42 regulates convergent extension movements during gastrulation through Wnt/Ca2+ signaling pathway. Dev Biol 2002;244:342-57.  Cockman M. The effects of folate deficiency on E-cadherin and β-catenin in colon epithelial cells. M.Sc. Thesis 2008. University of North Carolina at Greensboro.  Colapinto CK, O’Connor DL, Tremblay MS. Folate status of the population in the Canadian health measures survey. CMAJ 2011;183:E100-106.  Cole BF, Baron JA, Sandler RS et al. Folic acid for the prevention of colorectal adenomoas: A randomized clinical trial. JAMA 2007;297:2351-59.  Coombs GS, Yu J, Canning CA et al. Wls-dependent secretion of Wnt3a requires ser209 acylation and vacuolar acidification. J Cell Sci 2010;123:3357-67.  Cravo ML, Fidalgo P, Pereira AD et al. DNA methylation as an intermediate biomarker in colorectal cancer: modulation by folic acid supplementation. Eur J Cancer Prev 1994;3:473-79.  Cravo ML, Mason JB, Dayal Y et al. Folate deficiency enhances the development of colonic neoplasia in dimethylhydrazine-treated rats. Cancer Res 1992;52:5002-06.  Cravo ML, Pinto AG, Chaves P et al. Effect of folate supplementation on DNA methylation of rectal mucosa in patients with colonic adenomas: Correlation with nutrient intake. Clin Nutri 1998:17;45-49.  Crawford HC, Fingleton BM, Rudolph-Owen LA et al. The metalloproteinase matrilysin is a target of β-catenin transactivation in intestinal tumors. Oncogene 1999;18:2883-91.  Crott JW, Liu Z, Keyes MK et al. Moderate folate depletion modulates the expression of selected genes involved in cell cycle, intracellular signaling and folate uptake in human colonic epithelial cell lines. J Nutr Biochem 2008;19:328-35. 102   Cuskelly GJ, McNulty H, Scott JM. Effect of increasing dietary folate on red-cell folate: implications for prevention of neural tube defects. Lancet 1996;347:657-659.  Czeizel AE, Tímár L, Sárközi A. Dose-dependent effect of folic acid on the prevention of orofacial clefts. Pediatrics 1999;104:e66.  Das KC, Hoffbrand AV. Lymphocyte transformation in megaloblastic anaemia: Morphology and DNA synthesis. British Journal of Haematology 1970;19:459-468.   Das PM, Singal R. DNA methylation and cancer. J Clin Oncol 2004;22:4632-42.  De Wals P, Tairou F, Van Allen MI, et al. Reduction in neural-tube defects after folic acid fortification in Canada. N Engl J Med 2007;357:135-142.  Ding Q, Ko TC, Evers BM. Caco-2 intestinal cell differentiation is associated with G1 arrest and suppression of CDK2 and CDK4. Am J Cell Physiol 1998;275:C1193-C1200.  Duthie SJ, Narayanan S, Blum S et al. Folate deficiency in vitro induces uracil misincorporation and DNA hypomethylation and inhibits DNA excision repair in immortalized normal human colon epithelial cells. Nutrition and Cancer 2000;37:245-51.  Duthie SJ, Narayanan S, Brand GM et al. Impact of folate deficiency on DNA stability. J Nutr 2002;132:2444S-2449S.  Duval A, Gayet J, Zhou X et al. Frequent frameshift mutations of the TCF-4 gene in colorectal cancers with microsatellite instability. Cancer Res 1999;59:4213-15.  Ebbing M, Bønaa KH, Nygård O et al. Cancer incidence and mortality after treatment with folic acid and vitamin B12. JAMA 2009;302:2119-2126.  Fearnhead NS, Wilding JL, Winney B et al. Multiple rare variants in different genes account for multifactorial inherited susceptibility to colorectal adenomas. PNAS 2004;101:15992-97.  Figueiredo JC, Grau MV, Haile RW et al. Folic acid and risk of prostate cancer: Results from a randomized clinical trial. J Natl Cancer Inst 2009;101:432-435.  Freese JL, Pino D, Pleasure SJ. Wnt signaling in development and disease. Neurobiology of Disease 2010;38:148-53.  French MR, Barr SI, Levy-Milne R. Folate intakes and awareness of folate to prevent neural tube defects: A survey of women living in Vancouver, Canada. J Am Diet Assoc 2003;103:181-5.  103  Galli LM, Barnes TL, Secrest SS et al. Porcupine-mediated lipid-modification regulates the activity and distribution of Wnt proteins in the chick neural tube. Development 2007;134;3339-48.  Gelineau-van Waes J, Heller S, Bauer LK, et al. Embryonic development in the reduced folate carrier knockout mouse is modulated by maternal folate supplementation. Birth  Defects Research (Part A) 2008;82:494-507.  Giovannucci E, Stampfer MJ, Colditz GA et al. Multivitamin use, folate, and colon cancer in women in the Nurses’ Health Study. Annals of Internal Medicine 1998;129:517-24.  Glinka A, Wu W, Delius H et al. Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 1998;391:357-62.  Goldman ID. The characteristics of the membrane transport of amethopterin and the naturally occurring folates. Ann NY Acad Sci 1971;186:400-422.  Gonen N, Bram EE, Assarah YG. PCFT/SLC46A1 promoter methylation and restoration of gene expression in human leukemia cells. Biocehmical and Biophysical Research Communications 2008;376:787-792.  Greene NDE, Stanier P, Copp AJ. Genetics of human neural tube defects. Human Molecular Genetics 2009;18:R113-R129.  Hamblet NS, Lijam N, Ruiz-Lozano P et al. Dishevelled 2 is essential for cardiac outflow tract development, somite segmentation and neural tube closure. Development 2002;129:5827-38.  Harterink M, Korswagen HC. Dissecting the Wnt secretion pathway: key questions on the modification and intracellular trafficking of Wnt proteins. Acta Physiol 2012;204:8-16.  Harvey, S. The short-term bioavailability of microencapsulated folic acid and L-5-methyl-tetrahydrofolate. M.Sc. Thesis 2011. University of British Columbia.  He B, Reguart N, You L et al. Blockade of Wnt-1 signaling induces apoptosis in human colorectal cancer cells containing downstream mutations. Oncogene 2005;24:3054-58.  He T, Sparks AB, Rago C et al. Identificaiton of c-MYC as a target of the APC pathway. Science 1998;281:1509-12.  He T, Chan TA, Vogelstein B et al. PPARδ is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell 1999;99:335-45.  104  Health Canada. Dietary Reference Intakes: Reference Values for Vitamins. Nov 29, 2010. <http://www.hc-sc.gc.ca/fn-an/nutrition/reference/table/ref_vitam_tbl-eng.php> Retrieved Jan 2, 2011.  Health Canada. Canadian Community Health Survey Cycle 2.2, Nutrition. 2004. < http://www.hc-sc.gc.ca/fn-an/surveill/atlas/map-carte/index-eng.php#nu> Retrieved Jan 2, 2011.  Health Canada. Canadian Community Health Survey Cycle 2.2. Percentage of adults with a usual intake of folate below the estimated average requirement (EAR) in Canada. 2004. < http://www.hc-sc.gc.ca/fn-an/surveill/atlas/map-carte/adult_usu_folate-eng.php> Retrieved Jan 2, 2011.  Health Canada. Canadian Nutrient File. 2010. <www.hc-sc.gc.ca/fn-an/nutrition/fiche-nutri-data/index-eng.php> Retrieved Feb 19, 2013.  Herbert V. Studies of folate deficiency in man. Proceedings of the Royal Society of Medicine 1964;57:377-384.  Hirsch S, Sanchez H, Albala C et al. Colon cancer in Chile before and after the start of the flour fortification program with folic acid. Eur J Gastroenterol Hepatol 2009;21:436-439.  Hoang BH, Thomas JT, Abdul-Karim FW et al. Expression pattern of two Frizzled-related genes, Frzb-1 and Sfrp-1, during mouse embryogenesis suggests a role for modulating action of Wnt family members. Dev Dyn 1998;212:364-72.  Homocysteine Lowering Trialists’ Collaboration. Lower blood homocysteine with folic acid based supplements: meta-analysis of randomized trials. BMJ 1998;316:894-898.  Homocysteine Lowering Trialists’ Collaboration. Dose-dependent effects of folic acid on blood concentrations of homocysteine: a meta-analysis of the randomized trials. Am J Clin Nutr 2005;82:806-12.  Homocysteine Studies Collaboration. Homocysteine and risk of ischemic heart disease and stroke: A meta-analysis. JAMA 2002;288:2015-22.  HOPE-2 Investigators. Homocysteine lowering with folic acid and B vitamins in vascular disease. N Engl J Med 2006;354:1567-77.  Hseih J, Kodjbachian L, Rebbert ML et al. A new secreted protein that binds to Wnt proteins and inhibits their activities. Nature 1999;398:431-6.  Ikeda S, Kishida S, Yamamoto H et al. Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3β and β-catenin and promotes GSK-3β –dependent phosphorylation of β-catenin. EMBO J 1998;17:1371-84. 105   Ishitani T, Ninomiya-Tsuji J, Nagai S et al. The TAK1-NLK-MAPK-related pathway antagonize signalling between β-catenin and transcription factor TCF. Nature 1999;399:798-802.  Jacob RA. Folate, DNA methylation, and gene expression: Factors of nature and nurture. Am J Clin Nutr 2000;72:903-04.  Jacob RA, Gretz DM, Taylor PC et al. Moderate folate deplection increase plasma homocysteine and decreases lymphocyte DNA methylation in postmenopausal women. J Nutr 1998;128:1204-12.  Jansen G, Mauritz RM, Assaraf YG et al. Regulation of carrier-mediated transport of folates and antifolates in methotrexate-sensitive and –resistant leukemia cells. Advan Enzyme Regul 1997;37:59-76.  Jaszewski R, Millar B, Hatfield JS et al. Folic acid reduces nuclear translocation of β-catenin in rectal mucosal crypts of patients with colorectal adenomas. Cancer Letters 2004;206:27-33.  Jing M, Tactacan GB, Rodriguez-Lecompte JC et al. Proton-coupled folate transporter (PCFT): molecular cloning, tissue expression patterns and the effects of dietary folate supplementation on mRNA expression in laying hens. British Poultry Science 2010;51:635-38.  Johnson V, Volikos E, Halford SE et al. Exon 3 β-catenin mutations are specifically associated with colorectal carcinomas in hereditary non-polyposis colorectal cancer syndrome. Gut 2005;54:264-67.  Kamen BA, Smith AK. A review of folate receptor alpha cycling and 5-methyltetrahydrofolate accumulation with an emphasis on cell models in vitro. Advancde Drug Delivery Reviews 2004;56:1085-1097.  Kamen BA, Wang M, Streckfuss AJ et al. Delivery of folates to the cytoplasm of MA104 cells is mediated by a surface membrane receptor that recycles. J Biol Chem 1988;263:13602-13609.  Katanaev VL. The Wnt/Frizzled GPCR signaling pathway. Biochemistry (Moscow) 2010;75:1428-34.  Katula KS, Heinloth AN, Paules RS. Folate deficiency in normal human fibroblasts leads to altered expression of genes primarily linked to cell signaling, the cytoskeleton and extracellular matrix. J Nutr Biochem 2007;18:541-52.  Kawano Y, Kypta R. Secreted antagonists of the Wnt signalling pathway. J Cell Sci 2003;116:2627-34. 106   Keller R, Davidson LA, Shook DR. How we are shaped: The biomechanics of gastrulation. Differentiation2003;71:171-205.  Khosraviani K, Weir HP, Hamilton P et al. Effect of folate supplementation on mucosal cell proliferation in high risk patients for colon cancer. Gut 2002;51:195-99.  Kim DH, Smith-Warner SA, Spiegelman D et al. Pooled analyses of 13 prospective cohort studies on folate intake and colon cancer. Cancer Causes Control 2010;21:1919-1930.  Kim YI. Folate and carcinogenesis: Evidence, mechanisms, and implications. J Nutr Biochem 1999;10:66-88.  Kim YI. Folate, colorectal carcinogenesis, and DNA methylation: Lessons from animals studies. Environ Mol Mutagen 2004;44:10-25.  Kim YI. Nutritional epigenetics: Impact of folate deficiency on DNA methylation and colon cancer susceptibility. J Nutr 2005;135:2703-09.  Kim YI, Baik HW, Fawaz K et al. Effect of folate supplementation on two provisional molecular markers of colon cancer: A prospective, randomized trial. Am J Gastroenterol 2001;96:184-95.  Kim YI, Salomon RN, Graeme-Cook F et al. Dietary folate protects against the development of macroscopic colonic neoplasia in a dose responsive manner in rats. Gut 1996;39:732-40.  Komekado H, Yamamoto H, Chiba T et al. Glycosylation and palmitoylation of Wnt-3a are coupled ot produce an active form of Wnt-3a. Genes to Cells 2007;12:521-34.  Komiya Y, Habas R. Wnt signal transduction pathways. Organogenesis 2008;4:68-75.  Korinek V,  Barker N, Morin PJ et al. Consitutive transcriptional activation by a β-catenin-Tcf complex in APC-/- colon carcinoma. Science 1997;275:1784-87.  Koury MJ, Horne DW, Brown ZA, et al. Apoptosis of late-stage erythroblasts in megaloblastic anemia: Associate with DNA damage and macrocyte production. Blood 1997;89:4617-4623.  Krebs HA, Hems R, Tyler B. The regulation of folate and methionine metabolism. Biochem J 1976;158:341-353.  Kruh GD, Belinsky MG. The MRP family of drug efflux pumps. Oncogene 2003;22:7537-7552.  107  Kühl M, Sheldahl LC, Malbon CC et al. Ca2+/calmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus. J Biol Chem2000;275:12701-11.  Kurayoshi M, Yamamoto H, Izumi S et al. Post-translation palmitoylation and glycosylation of Wnt-5a are necessary for its signalling. Biochem J 2007;402:515-23.  Lamers Y, Prinz-Langenohl R, Bramswig S et al. Red blood cell folate concentrations increase more after supplementation with [6S]-5-methyltetrahydrofolate than with folic acid in women of childbearing age. Am J Clin Nutr 2006;84:156-61.  Lammi L, Arte S, Somer M et al. Mutations in AXIN2 cause familial tooth agenesis and predispose to colorectal cancer. Am J Hum Genet 2004;74:1043-50.  La Vecchia C, Negri E, Pelucchi C, et al. Dietary folate and colorectal cancer. Int J Cancer 2002;102:545-547.  Lee JE, Willett WC, Fuchs CS et al. Folate intake and risk of colorectal cancer and adenoma: modification by time. Am J Clin Nutr 2011;93:817-25.  Le Leu RK, Young GP, McIntosh GH. Folate deficiency reduces the development of colorectal cancer in rats. Carcinogenesis 2000;21:2261-65.  Lemos C, Kathmann I, Giovannetti E, et al. Folate deprivation induces BCRP (ABCG2) expression and mitoxantrone resistance in Caco-2 cells. Int J Cancer 2008;123:1712-1720.  Leppert M, Dobbs M, Scambler P et al. The gene for familial polyposis coli maps to the long arm of chromosome 5. Science 1987;238:1411-3.  Lin K, Wang S, Julius MA et al. The cysteine-rich frizzled domain of Frzb-1 is required and sufficient for modulation of Wnt signaling. PNAS 1997;94:11196-200.  Lindzon GM, Medline A, Sohn K et al. Effect of folic acid supplementation on the progression of colorectal aberrant crypt foci. Carcinogenesis 2009;30:1536-43.  Liu Y, Tomiuk S, Rozoy E et al. Thermal oxidation studies on reduced folate, L-5-methyltetrahydrofolic acid (L-5-MTHF) and strategies for stabilization using food matrices. J Food Sci 2012;77:C236-C243.  Liu Z, Choi SW, Crott JW et al. Mild depletion of dietary folate combined with other B vitamins alters multiple components of the Wnt pathway in mouse colon. J Nutr 2007;137:2701-08.  108  Loehrer FMT, Haefeli WE, Angst CP et al. Effect of methionine loading on 5-methyltetrahydrofolate, S-adenosylmethionine and S-adenosylhomocysteine in plasma of healthy humans. Clin Sci 1996;91:79-86.  Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 2004;20:781-810.  Lu W, Lin C, Roberts MJ et al. Niclosamide suppresses cancer cell growth by inducing Wnt co-receptor LRP6 degradation and inhibiting the Wnt/β-catenin pathway. PLoS ONE 2011;6:e29290. doi:10.1371/journal.pone.0029290  Lucas ML, Blair JA. The magnitude and distribution of the acid microclimate in proximal jejunum and its relation to luminal acidification. Proceedings of the Royal Society of London. Series B, Biological Sciences 1978;200:27-41.  Malaterre J, Ramsay RG, Mantamadiotis T. Wnt-Frizzled signalling and the many paths to neural development and adult brain homeostasis. Frontiers in Bioscience 2007;12:492-506.  Maliepaard M, Scheffer GL, Faneyte IF et al. Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Res 2001;61:3458-64.  Mann B, Gelos M, Siedow A et al. Target genes of β-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. PNAS 1999;96:1603-08.  Mason JB, Dickstein A, Jacques PF, et al. A temporal association between folic acid fortification and an increase in colorectal cancer rates may be illuminating important biological principles: A hypothesis. Cancer Epidemiol Biomarkers Prev 2007;16:1325-1329.  Maubon N, Le Vee M, Fossati L, et al. Analysis of drug transporter expression in human intestinal Caco-2 cells by real-time PCR. Fundamental & Clin Pharm 2007;21:659-663.  Melkonyan HS, Chang WC, Shapiro JP et al. SARPs: A family of secreted apoptosis-related proteins. PNAS 1997;94:13636-41.  Miwa N, Furuse M, Tsukita S et al. involvement of claudin-1 in the β-catenin/Tcf signaling pathway and its frequent upregulation in human colorectal cancers. Oncol Res 2001;12:469-76.  Mlodzik M. Planar cell polarization: Do the same mechanisms regulate Drosophila tissue polarity and vertebrate gastrulation? Trends Genet 2002;18:564-71.  109  Moon RT, Campbell RM, Christian JL et al. Xwnt-5A: a maternal Wnt that affects morphogenetic movements after overexpression in embryos of Xenopus laevis. Development 1993;119;97-111.  Moon RT, Kohn AD, De Ferrari GV et al. Wnt and β-catenin signalling: Diseases and therapies. Nature Reviews Genetics 2004;5:689-699.  Morillon II, YM. The effect of folate deficiency on the Wnt signalling pathway. M.Sc. Thesis 2008. University of North Carolina at Greensboro.  Morin PJ, Sparks AB, Kornek V et al. Activation of β-catenin-Tcf signaling in colon cancer by mutations in β-catenin or APC. Science 1997;275:1787-90.  Morris MS, Jacques PF, Rosenberg IH et al. Folate and vitamin B-12 status in relation to anemia, macrocytosis, and cognitive impairment in older Americans in the age of folic acid fortification. Am J Clin Nutr 2007;85:193-200.  MRC Vitamin Study Research Group. Prevention of neural tube defects: Results of the Medical Research Council Vitamin Study. Lancet 1991;338:131-137.  Munemitsu S, Albert I, Souza B et al. Regulation of intracellular β-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. PNAS 1995;92:3046-50.  Nakano H, Nakamura Y, Soda H et al. Methylation status of breast cancer resistance protein detected by methylation-specific polymerase chain reaction analysis is correlated inversely with its expression in drug-resistant lung cancer cells. Cancer 2008;112:1122-30.  Naya FJ, Mercer B, Shelton J et al. Stimulation of slow skeletal muscle fiber gene expression by calcineurin in vivo. JBC 2000;275:4545-48.  Nesbit CE, Tersak JM, Prochownik EV. MYC oncogenes and human neoplastic disease. Oncogene 1999;18:3004-16.  Ng X, Lucock M, Veysey M. Physicochemical effect of pH and antioxidants on mono- and triglutamate forms of 5-methyltetrahydrofolate, and evaluation of vitamin stability in human gastric juice: Implications for folate bioavailability. Food Chemistry 2008;106:200-10.  Ngamwongsatit P, Banada PP, Panbangred W et al. WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line. J Microbiological Methods 2008;73:211-15.  Novakovic P, Stempak JM, Sohn KJ et al. Effects of folate deficiency on gene expression in the apoptosis and cancer pathways in colon cancer cells. Carcinogenesis 2006;27:916-24. 110   Nusse R, Varmus HE. Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell 1982;31:99-109.  Nusslein-Volhard C, Wieschaus E. Mutations affecting segment number and polarity in Drosophila. Nature 1980;287:795-801.  Nyström M, Mutanen M. Diet and epigenetics in colon cancer. World J Gastroenterol 2009;15:257-263.  Običan SG, Finnell RH, Mills JL et al. Folic acid in early pregnancy: A public health success story. FASEB J 2010;24:4167-4174.  Patthy L. The WIF module. Trends Biochem Sci 2000;25:12-13.  Paulos CM, Reddy JA, Leamon CP, et al. Ligand binding and kinetics of folate receptor recycling in vivo: impact on receptor-mediated drug delivery. Mol Pharmacol 2004;66:1406-14.  Pellis L, Dommels Y, Venema D et al. High folic acid increases cell turnover and lowers differentiation and iron content in human HT29 colon cancer cells. British J Nutr 2008;99:703-08.  Pentieva K, McNulty H, Reichert R et al. The short-term bioavailabilities of [6S]-5-methyltetrahydrofolate and folic acid are equivalent in men. J Nutr 2004;134:580-85.  Pickhardt PJ, Choi JR, Hwang I et al. Nonadenomatous polyps at CT colongraphy: Prevalence, size distribution , and detection rates. Radiology 2004;232:784-90.  Piedrahita JA, Oetama B, Bennett GD, et al. Mice lacking the folic acid-binding protein Folbp1 are defective in early embryonic development. Nature Genetics 1999;23:228-232.  Polakis, P (2011). An introduction to Wnt signaling. In KH Goss and M Kahn (Eds.), Targeting the Wnt pathway in cancer (pp. 1-18). New York, NY: Springer  Science+Business Media.  Polakis P. The many ways of Wnt in cancer. Current Opinion in Genetics & Development 2007;17:45-51.  Prinz-Langenohl R, Brämswig S, Tobolski O et al. [6S]-5-methyltetrahydrofolate increases plasma folate more effectively than folic acid in women with the homozygous or wild-type 677C  T polymorphism of methylenetetrahydrofolate reductase. Br J  Pharmacol 2009;158:2014-21.  111  Public Health Agency of Canada. Evaluation of food fortification with folic acid for the primary prevention of neural tube defects. Dec 3, 2004. <http://www.phac-aspc.gc.ca/publicat/faaf/chap3-eng.php> Retrieved Jan 2, 2011.  Pufulete M, Al-Ghnaniem R, Khushal A et al. Effect of folic acid supplementation on genomic DNA methylation in patients with colorectal adenoma. Gut 2005;54:648-53.  Qiu A, Jansen M, Sakaris A et al. Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption. Cell 2006;127:917-928.  Rampersaud GC, Kauwell GP, Hutson AD et al. Genomic DNA methylation decreases in response to moderate folate depletion in elderly women. Am J Clin Nutr 2000;72:998-1003.  Reisenauer AM, Krumdieck CL, Halsted, CH. Folate conjugase: Two separate activities in human jejunum. Science 1977;198:196-197.  Rockman SP, Currie SA, Ciavarella M et al. Id2 is a target of the β-catenin/T cell factor pathway in colon carcinoma. JBC 2001;276:45113-19.  Ross JF, Chaudhuri PK, Ratnam M. Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines. Cancer 1994;73:432-443.  Rountree MR, Bachman KE, Herman JG et al. DNA methylation, chromatin inheritance, and cancer. Oncogene 2001;20:3156-65.  Sack U, Walther W, Scudiero D et al. S100A4-induced cell motility and metastasis is restricted by the Wnt/β-catenin pathway inhibitor calcimycin in colon cancer cells. Mol Biol Cell 2011;22:3344-54.  Said HM, Smith R, Redha R. Studies on the intestinal surface acid microclimate: developmental aspects. Pediatr Res 1987;22:497-499.  Sanjoaquin MA, Allen N, Couto E et al. Folate intake and colorectal cancer risk: A meta-analytical approach. Int J Cancer 2005;113:825-28.  Sansom OJ, Reed KR, Hayes AJ et al. Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration. Genes Dev 2004;18:1385-90.  Sansom OJ, Meniel VS, Muncan V et al. Myc deletion rescues Apc deficiency in the small intestine. Nature 2007;446:676-79.  Sauberlich HE, Kretsch MJ, Skala JH et al. Folate requirement and metabolism in nonpregnant women. Am J Clin Nutr 1987;46:1016-28.  112  Sauer H, Wilmanns W. Cobalamin dependent methionine synthesis and methyl-folate-trap in human vitamin B12 deficiency. Brit J Haematol 1977;36:189-98.  Selhub J, Morris MS, Jacques PF et al. Folate-vitamin B-12 interaction in relation to cognitive impairment, anemia, and biochemical indicators of vitamin B-12 deficiency. Am J Clin Nutr 2009;89:702S-706S.  Sharma RP, Chopra VL. Effect of the Wingless (wg1) mutation on wing and haltere development in Drosophila melanogaster. Dev Biol 1976;48:461-65.  Sheldahl LC, Park M, Malbon CC et al. Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner. Current Biology 1999;9:695-98.  Shen F, Ross JF, Wang X, et al. Identification of a novel folate receptor, a truncated receptor, and receptor type β in hematopoietic cells: cDNA cloning, expression,  immunoreactivity and tissue specificity. Biochemistry 1994;33:1209-1215.  Shimizu Y, Ikeda S, Fujimori M et al. Frequent alterations in the Wnt signaling pathway in colorectal cancer with microsatellite instability. Genes, Chromosomes & Cancer 2002;33:73-81.  Shuaibi AM, House JD, Sevenhuysen GP. Folate status of young Canadian women after folic acid fortication of grain products. J Am Diet Assoc 2008;108:2090-94.  Shtutman M, Zhurinsky J, Simcha I et al. The cyclin D1 gene is a target of the β-catenin/LEF-1 pathway. PNAS 1999;96:5522-27.  Sie KKY, Medline A, van Weel J et al. Effect of maternal and postweaning folic acid supplementation on colorectal cancer risk in the offspring. Gut 2011;60:1687-94.  Siegfried Z, Simon I. DNA methylation and gene expression. Syst Biol Med 2010;2:362-71.  Slusarski DC, Yang-Snyder J, Busa WB et al. Modulation of embryonic intracellular Ca2+ signaling by Wnt5a. Dev Biol 1997;182:114-120.  Smolich BD, McMahon JA, McMahon AP et al. Wnt family proteins are secreted and associated with the cell surface. Mol Biol Cell 1993;4:1267-75.  Solomon E, Voss R, Hall V et al. Chromosome 5 allele loss in human colorectal carcinomas. Nature 1987;328:616-9.  Song J, Medline A, Mason JB et al. Effects of dietary folate on intestinal tumorigenesis in the ApcMin mouse. Cancer Res 2000;60:5434-40.  113  Soucek L, Evan GI. The ups and downs of Myc biology. Current Opinion in Genetics & Development 2010;20:91-95.  Stampfer MJ, Manilow MR, Willet WC et al. A prospective study of plasma homocysteine and risk of myocardial infarction in US physicians. JAMA 1992;268:877-81.  Stewart AJ, Canitrot Y, Baracchini E, et al. Reduction of expression of the multidrug resistance protein (MRP) in human tumor cells by antisense phosphorothioate oligonucleotides. Biochem Pharmacol 1996;51:46-469.  Stierum R, Gaspari M, Dommels Y et al. Proteome analysis reveals novel proteins associated with proliferation and differentiation of the colorectal cancer cell line Caco-2. Biochimica et Biophysica Acta 2003;1650:73-91.  Su LJ, Arab L. Nutritional status of folate and colon cancer risk: Evidence from NHANES I epidemiologic follow-up study. Ann Epidemiol 2001;11:65-72.  Su L, Vogelstein B, Kinzler KW.  Association of the APC tumor suppressor protein with catenins. Science 1993;262:1734-37.  Subramanian VS, Chatterjee N, Said HM. Folate uptake in the human intestine: Promoter activity and effect of folate deficiency. J Cell Physiol 2003;196:403-08.  Subramanian VS, Reidling JC, Said HM. Differentiation-dependent regulation of the intestinal folate uptake process: Studies with Caco-2 cells and native mouse intestine. Am J Physiol Cell Physiol 2008;295:C828-35.  Suraweera N, Robinson J, Volikos E et al. Mutations within Wnt pathway genes in sporadic colorectal cancers and cell lines. Int J Cancer 2006;119:1837-42.  Suzuki H, Gabrielson E, Chen W et al. A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer. Nature Genetics 2002;31:141-49.  Suzuki H, Watkins DN, Jair KW et al. Epigenetic inactivation of SFRP genes allows constitutive Wnt signaling in colorectal cancer. Nature Genetics 2004;36:417-22.  Takada R, Satomi Y, Kurata T et al. Monounsaturated fatty acid modification of Wnt protein: Its role in Wnt secretion. Dev Cell 2006;11:791-801.  Tamai K, Semenov M, Kato Y et al. LDL-receptor-related proteins in wnt signal transduction. Nature 2000;407:530-35.  114  Tanaka K, Kitigawa Y, Kadowaki T. Drosophila segment polarity gene product porcupine stimulates the posttranslational N-glycosylation of wingless in the endoplasmic reticulum. JBC 2002;277:12816-23. Terry N, White RA. Flow cytometry after bromodeoxyuridine labeling to measure S and G2+M phase durations plus doubling times in vitro and in vivo. Nature Protocols  2006;1:859-869.  Tetsu O, McCormick F. β-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 1999;398:422-26.  Thwaites DT, Anderson CMH. H+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine. Exp Physiol 2007;92:603-619.  To KKW, Zhan Z, Bates SE. Aberrant promoter methylation of the ABCG2 gene in renal carcinoma. Mol Cell Biol 2006;26:8572-85.  Troen AM, Mitchell B, Sorensen B et al. Unmetabolized folic acid in plasma is associated with reduced natural killer cell cytoxicity among postmenopausaul women. J Nutr 2006;136:189-194.  Turner JG, Gump JL, Zhang C et al. ABCG2 expression, function, and promoter methylation in human multiple myeloma. Blood 2006;108:3881-89.  Ubbink JB, Hayward Vermaak WJ, van der Merwe A, et al. Vitamin requirements for the treatment of hyperhomocysteinemia in humans. J Nutr 1994;124:1927-1933.  Ulrich CM, Potter JD. Folate and cancer – Timing is everything. JAMA 2007;297:2408-09.  Ungar AR, Kelly GM, Moon RT. Wnt4 affects morphogenesis when misexpressed in the zebrafish embryo. Mech Dev 1995;52:153-64.  Van Den Heuvel M, Harryman-Samos C, Klingensmith J et al. Mutations in the segment polarity genes wingless and porcupine impair secretion of the wingless protein. EMBO J 1993;12:5293-5302.  van Rooij I, Ocké MC, Straatman H et al. Periconceptional folate intake by supplement and food reduces the risk of nonsyndromic cleft lip with or without cleft palate. Preventative Medicine 2004;39:689-694.  Veeman MT, Axelrod JD, Moon RT. A second canon: Functions and mechanisms of β-catenin-independent wnt signaling. Developmental Cell 2003;5:367-77.  Venn BJ, Green TJ, Moser R et al. Increased in blood folate indices are similar in women of childbearing age supplemented with [6S]-5-methyltetrahydrofolate and folic acid. J Nutr 2002;132 :3353-55. 115   Verlinde PHCJ, Oey I, Hendrickx ME et al. L-ascorbic acid improves the serum folate response to an oral dose of [6S]-5-methyltetrahydrofolic acid in healthy men. Eur J Clin Nutr 2008;62:1224-30.  Vilter RW, Will JJ, Wright T et al. Interrelationships of vitamin B12, folic acid and ascorbic acid in the megaloblastic anemias. J Clin Nutr 1963;12:130-144. .  Wang X, Fenech M. A comparison of folic acid and 5-methyltetrahydrofolate for  prevention of DNA damage and cell death in human lymphocytes in vitro. Mutagenesis 2003;18:81-86.  Wang S, Krinks M, Lin K et al. Frzb, a secreted protein expressed in the Spemann organizer, binds and inhibits Wnt-8. Cell 1997;88:757-66.  Wang Y, Nathans J. Tissue/planar cell polarity in vertebrates: new insights and new questions. Development 2007;134:647-58.  West Suitor C, Bailey LB. Dietary folate equivalents: Interpretation and application. J Am Diet Assoc. 2000;100:88-94.  Westfall TA, Brimeyer R, Twedt J et al. Wnt-5/pipetail function in vertebrate axis formation as a negative regulator of Wnt/β-catenin activity. J Cell Biol 2003;162:889-898.  Whetstine JR, Flatley RM, Matherly LH. The human reduced folate carrier gene is ubiquitously and differentially expressed in normal human tissues: identification of seven non-coding exons and characterization of a novel promoter. Biochem J 2002;367:629-640.  Wielenga VJ, Smits R, Korinek Vet al. Expression of CD44 in Apc and Tcf mutant mice implies regulation by the Wnt pathway. Am J Path 1999;154:515-23.  Willert J, Epping M, Pollack JR et al. A transcriptional response to Wnt protein in human embryonic carcinoma cells. BMC Dev Biol 2002;2:1-7.  Willert K, Brown JD, Danenberg E et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 2003;423:448-52.  Winkels RM, Brouwer IA, Siebelink E et al. Bioavailability of food folates is 80% of that of folic acid. Am J Clin Nutr 2007;85:465-73.  Woo KS, Chook P, Chan LLT et al. Long-term improvement in homocystein levels and arterial endothelial function after 1-year folic acid supplementation. Am J Med 2002;112:535-39.   116  Xia CQ, Liu N, Yang D et al. Expression, localization, and functional characteristics of breast cancer resistance protein in Caco-2 cells. Drug Metabolism and Disposition 2005;33:637-43.  Ying J, Li H, Yu J et al. WNT5A exhibits tumor-suppressive activity through antagonizing the Wnt/β-catenin signaling, and is frequently methylated in colorectal cancer. Clin Cancer Res 2008;14:55-61.  Zeng L, Fagotto F, Zhang T et al. The mouse Fused locus encodes axin, an inhibitor of the wnt signaling pathway that regulated embryonic axis formation. Cell 1997;90:181-192.  Zhai L, Chaturvedi D, Cumberledge, S. Drosophila wnt-1 undergoes a hydrophobic modification and is targeted to lipid rafts, a process that requires porcupine.  JBC 2004;279:33220-27.  Zhang T, Otevrel T, Gao Z et al. Evidence that APC regulates Survivin expression: A possible mechanism contributing to the stem cell origin of colon cancer. Cancer Res 2001;61:8664-67.  Zhang X, Gaspard JP, Chung DC. Regulation of Vascular Endothelial Growth Factor by the Wnt and K-ras pathways in colonic neoplasia. Cancer Res 2001;61:6050-54.  Zhao R, Matherly LH, Goldman ID. Membrane transporters and folate homeostasis: intestinal absorption and transport into systemic compartments and tissues. Expert Review in Molec Med 2009;11: e4.                      117  Appendices  Appendix A:  Flow Cytometry Diagrams      Histogram         Scatter Plot         Control 0.9 µM FA 2.3 µM FA 118         3.4 µM FA 0.9 µM 5M 2.3 µM 5M 119    Figure A.1: Cell cycle analysis of Caco2 cells after 3 days treatment with FA or 5MTHF. Caco2 cells were cultured in folate-free RPMI-1640 medium containing 0, 0.9, 2.3, or 3.4 µM FA or MTHF for 3 days. Cell cycle was determined using BrdU incorporation for S phase detection and PI staining for total DNA and analyzed by flow cytometry. The first column shows the histogram with percentage pre-G1 cells. The second column shows the corresponding scatter plot with the G1, S and G2/M phase gated and quantified. Abbreviations: FA (folic acid), 5M (5-methyltetrahydrofolate).               3.4 µM 5M 120  Histogram           Scatter Plot  Control 0.9 µM FA 2.3 µM FA 121    3.4 µM FA 0.9 µM 5M 2.3 µM 5M 122   Figure A.2: Cell cycle analysis of Caco2 cells after 5 days treatment with FA or 5MTHF. Caco2 cells were cultured in folate-free RPMI-1640 medium for 5 days and then transferred to folate-free RPMI-1640 medium containing 0, 0.9, 2.3, or 3.4 µM FA or 5MTHF for 5 days. Cell cycle was determined using BrdU incorporation for S phase detection and PI staining for total DNA and analyzed by flow cytometry. The first column shows the histogram with percentage pre-G1 cells. The second column shows the corresponding scatter plot with the G1, S and G2/M phase gated and quantified. Abbreviations: FA (folic acid), 5M (5-methyltetrahydrofolate).                           3.4 µM 5M 123  Appendix B:  Raw Data for β-Catenin Nuclear Localization  Table B.1 Raw Data for β-Catenin Nuclear Localization Sample Nucleus Fluorescence Nucleus Volume Nucleus (Fluor/vol) Whole Cell Fluorescence Whole Cell Volume Cytoplasmic Fluorescence Cytoplasmic Volume Cytoplasm (Fluor/vol) Adj. Nucleus: Cytoplasm 1-1-1a 97133246 10574 9186 529000000 67501 431866754 56926 7586 1.2 1-1-1b 87538356 9479 9235 485000000 67501 397461644 58022 6850 1.3 1-1-1c 109001374 11980 9099 443000000 56251 333998626 44271 7544 1.2 1-1-1d 71393268 8477 8422 404000000 56251 332606732 47774 6962 1.2 1-1-1e 147353877 16197 9098 512000000 67501 364646123 51304 7108 1.3 1-1-1f 119223099 14061 8479 523000000 67501 403776901 53440 7556 1.1         Average 1.2           1-1-2a 42966069 7943 5409 291000000 45001 248033931 37057 6693 0.8 1-1-2b 56282387 7756 7257 332000000 45001 275717613 37245 7403 1.0 1-1-2c 28156159 4082 6897 279000000 45001 250843841 40918 6130 1.1 1-1-2d 48549351 4725 10274 461000000 67501 412450649 62776 6570 1.6 1-1-2e 88135577 5767 15282 471000000 56251 382864423 50483 7584 2.0 1-1-2f 34767978 4840 7184 344000000 56251 309232022 51411 6015 1.2         Average 1. 3           1-1-3a 40615740 6703 6059 266000000 45001 225384260 38297 5885 1.0 1-1-3b 52570978 9392 5598 393000000 67501 340429022 58109 5858 1.0 1-1-3c 58812574 8696 6763 312000000 45001 253187426 36305 6974 1.0 1-1-3d 79078763 11979 6601 341000000 56251 261921237 44271 5916 1.1 1-1-3e 47873448 6468 7402 292000000 45001 244126552 38533 6336 1.2         Average 1.1           1-2-2a 17510091 3288 5326 226000000 45001 208489909 41713 4998 1.1 124  1-2-2b 134387549 14029 9579 528000000 67501 393612451 53472 7361 1.3 1-2-2c 97227575 15561 6248 464000000 67501 366772425 51940 7061 0.9 1-2-2d 30920913 6070 5094 230000000 45001 199079087 38931 5114 1.0 1-2-2e 46297279 6153 7525 399000000 67501 352702721 61348 5749 1.3         Average 1.1           1-2-3a 43058888 6958 6188 352000000 56251 308941112 49293 6267 1.0 1-2-3b 37966496 6032 6294 399000000 67501 361033504 61468 5873 1.1 1-2-3c 33937314 5382 6306 320000000 44998 286062686 39616 7221 0.9 1-2-3d 23956619 4141 5785 344000000 67501 320043381 63360 5051 1.1 1-2-3e 36768230 7221 5092 250000000 45001 213231770 37779 5644 0.9         Average 1.0           2-1-1a 31964606 5006 6385 401000000 67501 369035394 62494 5905 1.1 2-1-1b 60429838 12108 4991 253000000 56251 192570162 44142 4362 1.1 2-1-1c 49498520 9804 5049 305000000 67501 255501480 57696 4428 1.1 2-1-1d 68953110 13285 5190 381000000 78751 312046890 65466 4767 1.1 2-1-1e 33510717 6267 5347 304000000 67501 270489283 61234 4417 1.2         Average 1.1           2-1-2a 65797498 11793 5579 402000000 78751 336202502 66957 5021 1.1 2-1-2b 49381492 8426 5861 384000000 67501 334618508 59075 5664 1.0 2-1-2c 63679531 8627 7381 423000000 61369 359320469 52742 6813 1.1 2-1-2d 68051807 7582 8975 434000000 55868 365948193 48286 7579 1.2 2-1-2e 135341391 11004 12299 713000000 90001 577658609 78997 7312 1.7         Average 1.2           2-1-3a 58374483 10297 5669 400000000 67501 341625517 57204 5972 0.9 2-1-3b 60017663 8868 6768 362000000 67501 301982337 58633 5150 1.3 2-1-3c 24447966 3828 6386 303000000 56251 278552034 52422 5314 1.2 2-1-3d 35964677 5516 6521 361000000 67501 325035323 61985 5244 1.2 125  2-1-3e 56617398 6415 8825 368000000 67501 311382602 61085 5097 1.7         Average 1.3           2-2-1a 43412970 7365 5894 411000000 67501 367587030 60136 6113 1.0 2-2-1b 52863507 10341 5112 332000000 67501 279136493 57160 4883 1.0 2-2-1c 22266482 3697 6023 347000000 78751 324733518 75054 4327 1.4 2-2-1d 65895154 7718 8538 460000000 78751 394104846 71033 5548 1.5 2-2-1e 65867448 9076 7258 337000000 56251 271132552 47175 5747 1.3         Average 1.2           2-2-3a 66474840 7698 8636 482000000 78751 415525160 71053 5848 1.5 2-2-3b 50780335 7513 6759 374000000 67501 323219665 59988 5388 1.2 2-2-3c 47613029 6861 6939 425000000 67501 377386971 60640 6223 1.1 2-2-3d 51942751 9724 5342 323000000 67501 271057249 57777 4691 1.1 2-2-3e 26055651 3230 8066 325000000 56251 298944349 53020 5638 1.4         Average 1.3           3-1-2a 33898649 6332 5353 335000000 67501 301101351 61168 4922 1.1 3-1-2b 37248467 6669 5586 347000000 67501 309751533 60832 5092 1.1 3-1-2c 74346990 9983 7448 421000000 56251 346653010 46268 7492 1.0 3-1-2d 128519264 12627 10178 545000000 67501 416480736 54874 7590 1.3 3-1-2e 63750179 8527 7476 434000000 67501 370249821 58973 6278 1.2 3-1-2f 79799183 9336 8547 506000000 67501 426200817 58165 7327 1.2         Average 1.2           3-1-3a 23132478 4614 5014 270000000 56251 246867522 51637 4781 1.0 3-1-3b 64824532 13029 4975 420000000 87259 355175468 74229 4785 1.0 3-1-3c 49921658 9127 5469 388000000 78751 338078342 69623 4856 1.1 3-1-3d 80063738 11191 7154 499000000 90001 418936262 78810 5316 1.3 3-1-3e 38778888 6614 5863 378000000 67501 339221112 60887 5571 1.0         Average 1.1 126            3-2-1a 19362885 3068 6311 317000000 67501 297637115 64433 4619 1.4 3-2-1b 25776249 5446 4733 268000000 56251 242223751 50804 4768 1.0 3-2-1c 36113874 3632 9944 381000000 56251 344886126 52619 6554 1.5 3-2-1d 36913422 6930 5327 358000000 56251 321086578 49321 6510 0.8 3-2-1e 56728371 8594 6601 475000000 78751 418271629 70157 5962 1.1         Average 1.2           3-2-2a 21459899 3887 5520 313000000 67501 291540101 63613 4583 1.2 3-2-2b 20337504 3114 6530 343000000 67501 322662496 64387 5011 1.3 3-2-2c 38823178 5095 7620 332000000 67501 293176822 62406 4698 1.6 3-2-2d 52090974 6220 8375 391000000 78751 338909026 72531 4673 1.8 3-2-2e 128966570 21887 5892 429000000 67501 300033430 45614 6578 0.9         Average 1.4           3-2-3a 41291279 7328 5635 362000000 67501 320708721 60173 5330 1.1 3-2-3b 109701022 16361 6705 502000000 78751 392298978 62390 6288 1.1 3-2-3c 91488341 14537 6294 464000000 67501 372511659 52964 7033 0.9 3-2-3d 67593382 9457 7147 483000000 78751 415406618 69294 5995 1.2 3-2-3e 81426632 4802 16955 513000000 56251 431573368 51448 8388 2.0         Average 1.3           4-1-2a 45902675 9878 4647 310000000 67501 264097325 57623 4583 1.0 4-1-2c 62208312 11721 5307 395923863 89885 333715551 78163 4269 1.2 4-1-2d 113216079 22733 4980 339000000 67501 225783921 44768 5043 1.0 4-1-2e 61188503 10535 5808 392000000 67501 330811497 56966 5807 1.0 4-1-2f 29218432 5390 5421 326000000 67501 296781568 62111 4778 1.1         Average 1.1           4-1-3a 79220405 13771 5752 419000000 78751 339779595 64980 5229 1.1 4-1-3b 67608575 4518 14965 572000000 67501 504391425 62983 8008 1.9 127  4-1-3c 86804939 10868 7987 530000000 67501 443195061 56633 7826 1.0 4-1-3d 46344823 9101 5092 364000000 78751 317655177 69650 4561 1.1 4-1-3e 49148420 8489 5789 418000000 78751 368851580 70262 5250 1.1 4-1-3f 36515749 6773 5391 329000000 56251 292484251 49477 5911 0.9         Average 1.2           4-2-1a 65680083 10963 5991 399000000 78751 333319917 67788 4917 1.2 4-2-1b 113331551 19156 5916 490000000 78748 376668449 59592 6321 0.9 4-2-1c 50865453 6224 8173 362000000 67501 311134547 61277 5077 1.6 4-2-1d 32768577 5145 6369 292000000 56251 259231423 51106 5072 1.2 4-2-1e 56554405 5632 10041 455000000 67501 398445595 61869 6440 1.6         Average 1.3           4-2-2a 55111779 6497 8483 443000000 67501 387888221 61004 6358 1.3 4-2-2b 97309509 15588 6243 442000000 78751 344690491 63163 5457 1.1 4-2-2c 21156893 2094 10104 327000000 56251 305843107 54157 5647 1.8 4-2-2d 40173567 4688 8569 386000000 67501 345826433 62813 5506 1.6 4-2-2e 58082212 9837 5904 379000000 67501 320917788 57664 5565 1.1         Average 1.4           5-1-2a 92246842 8755 10537 626000000 90001 533753158 81246 6570 1.6 5-1-2b 86230894 7100 12145 698000000 90001 611769106 82901 7380 1.6 5-1-2d 198893991 22512 8835 882000000 101251 683106009 78739 8676 1.0 5-1-2e 103314251 13648 7570 635000000 78751 531685749 65103 8167 0.9 5-1-2f 207932335 29081 7150 790000000 90001 582067665 60920 9555 0.7         Average 1.2           5-1-3b 66105310 3263 20260 519000000 56251 452894690 52988 8547 2.4 5-1-3c 72770074 5294 13745 605000000 56251 532229926 50956 10445 1.3 5-1-3d 325740169 21021 15496 1100000000 78751 774259831 57730 13412 1.2 5-1-3e 121972571 7744 15750 776000000 67501 654027429 59756 10945 1.4 128  5-1-3f 89055720 7846 11351 570000000 56251 480944280 48405 99356 1.1         Average 1.5           5-2-2a 90362686 5098 17724 693000000 45001 602637314 39902 151023 1.2 5-2-2b 83116484 11223 7406 439000000 67501 355883516 56278 6324 1.2 5-2-2c 47756214 2982 16017 427000000 56251 379243786 53269 7119 2.2 5-2-2d 78714269 8058 9769 426000000 56251 347285731 48193 7206 1.4 5-2-2e 50761821 3048 16652 421000000 56251 370238179 53202 6959 2.4 5-2-2f 50556755 4626 10930 392000000 56251 341443245 51625 6614 1.6 5-2-2g 80083076 10146 7893 412000000 56250 331916924 46104 7199 1.1 5-2-2h 46460343 6794 6838 355000000 56251 308539657 49456 6239 1.1         Average 1.5           6-1-1a 50078320 6422 7798 290000000 56251 239921680 49829 4815 1.6 6-1-1b 94156045 12222 7704 570000000 78751 475843955 66529 7152 1.1 6-1-1c 60275432 5677 10616 414000000 67501 353724568 61823 5722 1.9 6-1-1d 43623927 4449 9805 417000000 67501 373376073 63052 5922 1.7 6-1-1e 81736180 7452 10969 526000000 67501 444263820 60049 7398 1.5         Average 1.6           6-1-2a 50670187 6872 7373 447000000 78751 396329813 71879 5514 1.3 6-1-2b 102664729 9033 11365 441000000 56251 338335271 47217 7165 1.6 6-1-2c 39833820 6079 6552 358000000 67501 318166180 61421 5180 1.3 6-1-2d 81106521 9559 8484 537000000 78751 455893479 69192 6589 1.3 6-1-2e 59775928 9963 6000 329000000 56251 269224072 46287 5816 1.0         Average 1.3           6-1-3a 92270956 9755 9458 568000000 787501 475729044 68996 6895 1.4 6-1-3b 98876988 18179 5439 418000000 78751 319123012 60571 5268 1.0 6-1-3c 53566927 9714 5514 283000000 56251 229433073 46537 4930 1.1 6-1-3d 26955735 5071 5315 368000000 78751 341044265 73680 4629 1.1 129  6-1-3e 48122576 7557 6368 427000000 67501 378877424 59944 6320 1.0         Average 1.1           6-2-1a 89899710 7318 12285 692000000 90001 602100290 82683 7282 1.7 6-2-1b 55573194 7413 7497 484000000 78751 428426806 71338 6006 1.2 6-2-1c 88334931 10636 8305 759000000 101251 670665069 90615 7401 1.1 6-2-1d 80077390 4959 16147 613000000 78751 532922610 73792 7222 2.2 6-2-1e 41686957 5756 7243 381000000 67501 339313043 61745 5495 1.3         Average 1.5           6-2-3a 33150850 5854 5663 312000000 67501 278849150 61647 4523 1.2 6-2-3b 46717347 7800 5990 356000000 67501 309282653 59701 5180 1.2 6-2-3c 54882526 7656 7168 389000000 67501 334117474 59844 5583 1.3 6-2-3d 56096138 7384 7597 467000000 67501 410903862 60116 6835 1.1 6-2-3e 48639256 7260 6700 308000000 56251 259360744 48991 5294 1.3         Average 1.2           7-1-1a 42386135 7452 5688 362000000 67501 319613865 60048 5323 1.1 7-1-1b 173152651 27341 6333 593000000 101251 419847349 73910 5680 1.1 7-1-1c 34081518 4729 7207 338000000 56251 303918482 51521 5899 1.2 7-1-1d 43408291 6943 6252 431000000 67501 387591709 60557 6400 1.0 7-1-1e 65195846 7051 9246 411000000 67501 345804154 60450 5720 1.6         Average 1.2           7-1-2a 20800308 3260 6381 298000000 56251 277199692 52991 5231 1.2 7-1-2b 71746390 12215 5873 371000000 67501 299253610 55285 5413 1.1 7-1-2c 28770018 4210 6834 340000000 67501 311229982 63291 4917 1.4 7-1-2d 57566914 9622 5982 368000000 67501 310433086 57878 5364 1.1 7-1-2e 49654506 6422 7732 406000000 67501 356345494 61079 5834 1.3         Average 1.2           130  *Data is represented in Figure 2.8 and 2.9 Sample identity is organized by folate treatment (1=control, 2=0.9 µM FA, 3=2.3 µM FA, 4=3.4 µM FA, 5=0.9 µM 5MTHF, 6=2.3 µM 5MTHF, 7=3.4 µM 5MTHF), slide number (1-2), position on slide (1- 3), and locations sampled within a sample (a-h). 7-1-3a 38166885 4459 8560 387000000 67501 348833115 63042 5533 1.5 7-1-3b 24717780 4269 5791 291000000 56251 266282220 51982 5123 1.1 7-1-3c 31498136 5390 5844 320000000 67501 288501864 62111 4645 1.3 7-1-3d 77286769 8784 8799 466000000 67501 388713231 58717 6620 1.3 7-1-3e 23662420 4022 5883 317000000 67501 293337580 63479 4621 1.3         Average 1.3           7-2-1a 88045587 10300 8548 521000000 78751 432954413 68451 6325 1.4 7-2-1b 61850907 8116 7621 431000000 67501 369149093 59385 6216 1.2 7-2-1c 56192294 9971 5636 359000000 67501 302807706 57530 5263 1.1 7-2-1d 43578441 6949 6271 377000000 78751 333421559 71802 4644 1.4 7-2-1e 112884890 18472 6111 598000000 101251 485115110 82779 5860 1.0         Average 1.2 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0167325/manifest

Comment

Related Items