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In Vitroand in Vivo Studies of Antioxidant and Anti-Breast

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In Vitroand in Vivo Studies of Antioxidant and Anti-Breast IN VITRO AND IN VIVO STUDIES OF ANTIOXIDANT AND ANTI-BREAST CANCER ACTIVITIES OF POMIFERIN A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph By RAYMOND X. YANG In partial fulfilment of requirements for the degree of Doctor of Philosophy December, 2008 © Raymond Yang, 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-50154-2 Our file Notre reference ISBN: 978-0-494-50154-2 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada ABSTRACT IN VITRO AND IN VIVO STUDIES OF ANTIOXIDANT AND ANTI-BREAST CANCER ACTIVITIES OF POMIFERIN Raymond X. Yang Advisor: University of Guelph, 2008 Dr. Kelly Meckling Co-advisor: Dr. Rong Cao Reactive oxygen species (ROS) are ubiquitous in the human body and play a pivotal role in many chronic diseases, including cancer. The human body has developed a strong antioxidant defense system. Unfortunately, this system cannot be adequately maintained with ageing. The supply of exogenous antioxidants to help the body fight these diseases is a logical approach. Polyphenols, particularly flavonoids, have been found to be strong antioxidants. In this study, the prenylated isoflavone, pomiferin, from Osage orange, was examined. I found that pomiferin had a much stronger antioxidant activity than soy isoflavones, genistein and daidzein, in three different antioxidant assays including P-CLAMS, FRAP and PCL. By using both estrogen receptor positive (MCF-7) and negative (MDA-MB-435) cancer cell lines, pomiferin showed much lower IC50 values than soy isoflavones in both cell lines (5.2 ± 0.90 vs 5.4 ± 0.72 uM) after 24 hr treatment. The selectivity of pomiferin between cancer cells and MCF-10A (spontaneously immortalized human breast epithelial cell line) was high. Microarray techniques were used to find the gene expression changes when all three cells were treated with 5.0 uM pomiferin. At p <0.05, 515, 691 and 59 genes were significantly regulated for MCF-7, MDA-MB-435 and MCF-10A, respectively; and at p < 0.01 cut off, 94, 105 and 1 genes, respectively. Many of these genes are associated with antioxidant enzymes, cell cycle regulation and apoptosis. To confirm the results from microarray, RT-qPCR was used to verify some of the gene expression changes. The expression of 20 out of 21 genes was confirmed. In addition, the xenograft models of MCF-7 and MDA-MB-435 cells were established to find the in vivo anticancer activity of pomiferin. The tumour size was significantly reduced in MCF-7 with 0.2% and MDA-MB-435 with 0.5% of pomiferin in their diet (p < 0.05). Bioavailability of pomiferin was measured and the correlation with tumour sizes was evaluated. Plasma pomiferin level can partially explain the tumour size, but many other factors likely play a role. Further studies are needed to determine the specific mechanism(s) by which pomiferin alters specific gene expression and the differential effects in tumour versus normal cells. ACKNOWLEDGEMENTS I would like to extend my sincere thanks and appreciation to my supervisors, Drs Rong Cao and Kelly Meckling. Throughout my work and study, you have continuously served as a catalyst for novel scientific thought and supported my scientific pursuits. It is because of your guidance that I decided to pursue a PhD degree, and it is because of your guidance that I have succeeded. I am indebted to you more than words could possibly express. I would also like to thank my advisor, Dr Brenda Coomber, who has given me invaluable and critical advice through my study. You are always kind, accessible and highly professional. You have impressed me a lot. I acknowledge the assistance and wonderful friendship from my colleagues and students in both Drs Cao's and Meckling's laboratories. Special thanks to Drs Xinhua Yin, Hai Yu, Joy Liu, Suqin Shao and Cynthia Richard, Ms Honghui Zhu and the last, but not the least, Jane He. I also greatly acknowledge the friendly and thorough assistance from Jing Zhang at the University Microarray Facility, who spent hours and hours work to help and train me for microarray techniques and data processing. I also thank Dr Jiping Li and Honghong Li from the Laboratory Service Division to help set up RT-qPCR experiment, Dr. Jun Gu from University NMR Facility in helping run all the samples, and Jackie Rombeek from the Central Animal Facility of University of Guelph to take good care of my mice. This work would not have been possible without the constant encouragement and support from my wife, Amy, my children, Ray and Ethan, and my extended family. I appreciate their patience and tolerance during the course of my study. TABLE OF CONTENTS ABSTRACTS TABLE OF CONTENTS i LIST OF TABLES iv LIST OF FIGURES v ABBREVIATIONS vi CHAPTERI LITERATURE REVIEW 1.1 Introduction 1 1.2 Sources of ROS 2 1.2.1 Mitochondria 2 1.2.2 NADPH oxidases 4 1.2.3 Xanthine oxidase 5 1.2.4 Cytochrome P-450 5 1.2.5 Microsomes and peroxisomes 6 1.2.6 Transition Metals 6 1.3 ROS and biological macromolecules 8 1.3.1 ROS and lipid peroxidation 8 1.3.2 ROS and protein oxidation 9 1.3.3 ROS and DNA modifications 9 1.4 ROS and processes of carcinogenesis 10 1.5 Antioxidants 12 1.5.1 Enzymatic Antioxidants 13 1.5.1.1 SOD 14 1.5.1.2 GPx 14 1.5.1.3 CAT 16 1.5.2 Non-enzymatic antioxidants 16 1.5.2.1 GSH 17 1.5.2.2 TRX 18 1.5.2.3 NADPH &G6PH 18 1.5.3 Flavonoids 20 1.5.3.1 Inhibition of pro-oxidant enzymes 20 1.5.3.2 Metal Chelating effects 22 1.5.3.3 Enhancement and protection of antioxidant enzymes 23 1.5.3.4 Strong antioxidants 23 1.5.3.5 Direct binding 24 1.5.3.6 Phytoestrogens & anti-aromatase activity 24 1.5.3.7 Cell cycle regulation 25 1.5.3.8 Anti-Angiogenesis and Anti-Metastasis activity 26 1 1.5.3.9 Apoptosis 26 1.6 Epidemiological study of flavonoids and cancer 27 1.7 Hypothesis 28 CHAPTER II CHEMISTRY AND ANTIOXIDANT ACTIVITY OF PRENYLATED ISOFLAVONES FROM OSAGE ORANGE 2.1 Introduction 30 2.2 Materials and Methods 32 2.2.1 Chemicals and Solvents 33 2.2.2 Extraction and Purification of Isoflavones 35 2.2.3 Structure Identification with MS and NMR 36 2.2.4 Quantification of Osajin and Pomiferin 36 2.2.5 Antioxidant assays 37 2.2.5.1 Ferric Reducing/Antioxidant Power Assay 37 2.2.5.2 P-Carotene-Linoleic Acid Model System 38 2.2.5.3 Photochemiluminescent Assay 38 2.2.6 Statistics 39 2.3 Results 39 2.3.1 Structural confirmation 3 9 2.3.2 Yield and purity of pomiferin and osajin 44 2.3.3 Antioxidant activity 44 2.4 Discussion 49 CHAPTER III ANTIPROLIFERATIVE EFFECTS OF POMIFERIN IN MCF-7 AND MDA-MB-435 BREAST CANCER CELL 3.1 Introduction 53 3.2 Materials and Methods 55 3.2.1 Cell cultures and Proliferation assays 5 5 3.2.1.1 Cell Culture 55 3.2.1.2 Proliferation assays 56 3.2.1.3 Sulforhodamine B (SRB) dye-binding assay 56 3.2.1.4 Calculations of IC50 values 57 3.2.2 RNA extraction 57 3.2.3 Microarray 59 3.2.3.1 Preparation of array slides 59 3.2.3.2 Scanning and Data Processing 60 3.2.4 RT-qPCR 61 3.3 Results 64 3.3.1 Antiproliferative Activity of Pomiferin 64 3.3.2 Quality and Quantity of RNA 66 3.3.3 Microarray Anaysis 69 3.3.4 RT-qPCR study 73 3.3.5 Selected pathways associated with MCF-7 genes 76 3.3.5.1 TGF-p Signaling Pathway 76 3.3.5.2 Cell cycle regulation 79 11 3.3.6 The functions of the genes in MCF-7 confirmed by RT-qPCR 83 3.3.6.1 MnSOD 83 3.3.6.2 GPX3 84 3.3.6.3 TXNRD1 85 3.3.6.4 FTL 86 3.3.6.5 HSPA1A 87 3.3.6.6 HMGB1 87 3.3.6.7 TOP2A 88 3.3.6.8 PSMA5 89 3.3.6.9 CANXand 5G4P57 90 3.3.6.10 C/Z5P 91 3.3.6.11 ffi^FJ 92 3.3.7 Biological functions and pathways associated with MDA-MB-435 genes 92 3.3.8 The functions of the MDA-MB-435 genes confirmed by RT-qPCR 93 3.3.8.1 DICER1 93 3.3.8.2 ABCE1 94 3.3.8.3 TFE3 94 3.3.8.4 MFN2 95 3.3.S.5S100P 96 3.3.9 Pomiferin and Melanoma 97 3.4 Discussion 97 CHAPTER IV ANTITUMOUR ACTIVITY OF POMIFERIN IN XENOGRAFT MODLES OF HUMAN BREAST CANCERS 4.1 Introduction 101
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