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The Investigation of the Effects of Metformin in Thyroid Cancer

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The Investigation of the Effects of Metformin in Thyroid Cancer The investigation of the effects of Metformin in Thyroid Cancer By: Safar Kheder A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Supervisors: Saba Balasubramanian, Karen Sisley and Sirwan Hadad The University of Sheffield Department of Oncology and Metabolism Medical School Sheffield, UK. February 2018 1 Acknowledgement This is my pleasure to take this valuable opportunity to show my immense gratitude to my supervisors Dr. Saba Balasubramanian, Dr. Karen Sisley and Dr. Sirwan Hadad for their patience, support, enthusiasm, motivation, and important help during my PhD. I could not expected to have better supervision during my PhD. Dr. Saba Balasubramanian is very welcoming and has spent time with me during and beyond office hours. He is keen to give me advice and guides me in the right direction with my endless enquiry and questions throughout my PhD. Dr. Karen Sisley is always keen to help me alongside my laboratory work in the lab or her office and she has trained me very well during my PhD. Dr. Sirwan Hadad enriched me with his great knowledge on Metformin as an anti-cancer agent and guided me with his excellence supervision. I would like to thank several people (David Hammond, Claire Greaves, Azeez Salawu, Nawal Al Shammari, Afnan Al Kathiri, Meshal AlHaji Mohammed, Shamsa Ihmed, Dler Kadir) that they have important role in the success of my PhD. Human Capacity Development Program (HCDP) in Kurdistan Regional Government-Iraq (KRG) sponsored this work and I want to say this work was impossible to complete it without their funds and support. Finally, I would like to take this opportunity to thank my family for their understanding and unconditional support throughout my PhD. 2 Abstract Background: Thyroid cancer is generally associated with an excellent prognosis, but there is significant long-term morbidity with standard treatment, and some sub-types have poor prognosis. Metformin, an oral anti-diabetic drug is shown to have anti-cancer effects in several types of cancer (breast, lung and ovarian cancer). Proposed mechanisms include activation of Adenosine Mono-phosphate-activated Protein Kinase (AMPK) pathway; inhibition of mTOR pathway; reduction in blood glucose and insulin levels by inhibiting hepatic gluconeogenesis and increasing peripheral glucose uptake. OCT1 (organic cation transporter 1) helps in the uptake of Metformin into liver cells. Aims: Explore the anti-cancer effect of Metformin on the growth and proliferation of thyroid cancer cell lines. Methods: The effects of Metformin on thyroid cancer cell lines (FTC-133, K1E7, RO82-W-1, 8305C and TT) and normal thyroid follicular cells (Nthy-ori 3-1) were investigated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay for cell proliferation; clonogenic assay; Fluorescence-activated Cell Sorting (FACS) analysis for apoptosis (using double staining with Annexin V-FITC stained early apoptotic cells and propidium iodide stained late apoptotic cells) and cell cycle (using propidium iodide staining); and H2A.X phosphorylation (γH2AX) assay for DNA repair. Immunocytochemistry was used to investigate OCT1 expression to evaluate the mechanism of action of Metformin. The effects of Thyroid Stimulating Hormone (TSH), insulin and Insulin-like Growth Factor- 1 (IGF-1) on the response to Metformin treatment were also investigated. Affymetrix assay was used for gene expression profiling of cell lines before and after Metformin treatment. Results: Metformin inhibited cell proliferation and colony formation at a minimum concentration of 0.03 mM and increased the percentage of apoptotic 3 cells at concentrations of 0.1 mM and above. Metformin also induced cell cycle arrest in G0/G1 phase at minimum concentration of 0.3 mM. Unlike previous reports, Metformin did not appear to affect response to DNA repair. OCT1 expression was observed in all thyroid cancer cell lines, but no significant difference was observed in the proportion and intensity of OCT1 expression in Metformin treated and non-treated cells. Anti-cancer effects of Metformin (cell proliferation and apoptosis) was amplified in the glucose-free medium compared to glucose-rich medium. Metformin inhibited cell proliferation regardless of the presence of TSH, IGF-1 and insulin in the medium. Metformin modulated the expression of numerous genes and provided clues to the mechanism of action. Conclusion: Metformin suppresses thyroid cancer proliferation in vitro at concentrations within the therapeutic range for diabetic patients. This effect appears to be independent of TSH, insulin, glucose and IGF-1. Further work is needed to determine the molecular mechanisms underlying the observed effects. 4 Publications Research article: Kheder S, Sisley K, Hadad S, Balasubramanian SP., et al., effects of prolonged exposure to low dose Metformin in thyroid cancer. J Cancer, 2017. 8(6): p. 1053-1061. Poster and oral presentation: Kheder S, Sisley K, Hadad S, Balasubramanian SP the in vitro effects of Metformin on thyroid cancer cell lines at the 15th International Thyroid Congress, Florida 2015 Poster presentation: Kheder S, Sisley K, Hadad S, Greaves C, Balasubramanian SP importance of the OCT1 transporter to the anti-cancer effect of Metformin in thyroid cancer, presented at the 11th Annual School Research Meeting 2015, The Medical School, University of Sheffield, UK; and 35th BAETS Annual Scientific Meeting Reading/ Henley on Thames 2015 Oral presentation: Kheder S, Sisley K, Hadad S, Balasubramanian SP investigation of the role of Metformin in thyroid cancer presented at the BJS Prize session of the 34th BAETS Annual Scientific Meeting in Liverpool 2014. European Journal of Surgical Oncology (EJSO), Volume 40, Issue 12, December 2014, Pages 1799-1800 http://dx.doi.org/10.1016/j.ejso.2014.07.010http://dx.doi.org/10.101 6/j.ejso.2014.07.010 Poster presentation: Kheder S, Sisley K, Hadad S, Balasubramanian SP investigation of the role of Metformin in thyroid cancer, presented at the 10th Annual Research Meeting 2014, The Medical School, University of Sheffield, UK. 5 Table of contents 1 Chapter one .................................................................................................. 18 1.1 Introduction .................................................................................................... 19 1.2 Thyroid Gland ................................................................................................. 19 1.2.1 Thyroid Gland Anatomy ........................................................................... 19 1.2.2 Thyroid Gland Physiology ........................................................................ 20 1.2.3 Thyroid Diseases ..................................................................................... 21 1.3 Metformin ....................................................................................................... 36 1.3.1 Metformin structure .................................................................................. 36 1.3.2 Metformin indications dose and side effects............................................. 36 1.3.3 Pharmacokinetics of Metformin ................................................................ 37 1.3.4 Mechanism of action of Metformin ........................................................... 38 1.3.5 Possible role of Metformin in Cancer ....................................................... 43 1.3.6 Metformin and its effect on TSH ............................................................... 51 1.4 Need for this research and implications for patients ........................................ 53 2 Chapter two .................................................................................................. 55 2.1 Materials ......................................................................................................... 56 2.1.1 Basic laboratory equipment ..................................................................... 56 2.1.2 Other materials ........................................................................................ 57 2.1.3 Cell lines and medium ............................................................................. 59 2.2 Methods ......................................................................................................... 60 2.2.1 Cell culture .............................................................................................. 60 2.2.2 Cell proliferation assay (MTT assay) ........................................................ 64 2.2.3 Cell proliferation assay (trypan blue assay) ............................................. 66 2.2.4 Clonal formation assay ............................................................................ 68 2.2.5 Cell migration or scratch assay ................................................................ 69 2.2.6 Flow cytometric analysis for apoptosis assay ........................................... 70 2.2.7 Flow cytometric analysis for cell cycle assay ........................................... 72 2.2.8 γH2AX assay for DNA repair .................................................................. 73 2.2.9 Immunocytochemistry for OCT1 expression ............................................ 76 2.2.10 Affymetrix assay .................................................................................... 78 2.2.11 Statistical analyses ................................................................................ 79 3 Chapter three ...............................................................................................
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