SMALL MOLECULE GROWTH INHIBITORS FROM Albatrellus flettii AND Sarcodon scabripes NATIVE TO BRITISH COLUMBIA by Almas Yaqoob Doctor of Pharmacy (Pharm-D), Hamdard University, Pakistan, 2012 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOCHEMISTRY UNIVERSITY OF NORTHERN BRITISH COLUMBIA April 2019 © Almas Yaqoob, 2019 Abstract The first part of this thesis investigated the growth-inhibitory and immunomodulatory potential of six wild Canadian mushrooms. Out of 24 crude extracts, six showed strong growth- inhibitory activity, two exhibited strong immuno-stimulatory activity and nine demonstrated potent anti-inflammatory activity. The second part of this thesis involved purification and characterization of growth- inhibitory compounds from Albatrellus flettii. Liquid-liquid extraction, Sephadex LH-20 and HPLC-Mass Spectrometry (HPLC-MS) were used to purify the three compounds of interest. NMR analyses confirmed their identity as grifolin, neogrifolin and confluentin. Grifolin and neogrifolin inhibited IMP1-KRas RNA interaction as demonstrated using an in-vitro fluorescent polarization assay. The three compounds suppressed KRas expression in SW480 and HT-29 human colon cancer cells. Confluentin, shown for the first time, to induce apoptosis and arrest cell cycle in SW480 cells. The third part of this thesis involved the development of methods to purify growth-inhibitory compounds from Sarcodon scabripes. HPLC-MS detected some potential novel compounds. ii Table of contents Abstract ........................................................................................................................................... i List of Tables .............................................................................................................................. viii List of Figures ............................................................................................................................... ix List of Abbreviations ................................................................................................................. xvi Acknowledgements ................................................................................................................... xvii Chapter 1: Introduction and literature review .......................................................................... 1 1.1 Mushrooms are not just food ............................................................................................................ 1 1.2 Mushrooms as ancient remedy ......................................................................................................... 2 1.3 Medicinal mushrooms and their compounds as a source of anti-proliferative activity .................. 2 1.3.1 Anti proliferative mushrooms ...................................................................................................... 3 1.3.2 Cytotoxic Extracts ......................................................................................................................... 4 1.4 Overall Medicinal Prospects of Mushrooms ..................................................................................... 5 1.4.1 Anti-Bacterial Mushrooms ........................................................................................................... 5 1.4.2 Cardiovascular diseases ............................................................................................................... 6 1.4.3 Anti-inflammatory ........................................................................................................................ 6 1.4.4 Anti-oxidative ............................................................................................................................... 7 1.5 Wild mushrooms in Canada: a source to discover new anti-cancer compounds ............................ 7 1.6 Compounds with anti-proliferative activity ...................................................................................... 9 1.6.1 Large molecules (High Molecular weight compounds) ............................................................. 10 1.6.2 Small molecules (Low Molecular weight compounds) .............................................................. 12 1.7 Research Goals ................................................................................................................................. 14 Chapter 2: Evaluation of Canadian wild mushrooms for growth-inhibitory and immuno- modulatory activities .................................................................................................................. 15 2.1 Materials and Methods ......................................................................................................... 15 2.1.1 Mushroom Collection & Identification ......................................................................................... 15 2.1.2 Genetic identification of the mushroom samples using ITS3 and NLB4 primers ........................ 18 2.1.3 DNA extraction procedures .......................................................................................................... 19 2.1.4 PCR amplification and purification procedure ............................................................................. 19 2.2 Chemical extraction of mushrooms ..................................................................................... 21 2.2.1 Rotary evaporation, pH neutralization, snake skin dialysis, and lyophilization of the extracts 22 2.2.2 Stock solution and filter sterilization ........................................................................................... 23 iii 2.3 Assessment of Anti-proliferative and Immuno-Modulatory Activities (Immuno- stimulatory and Anti-inflammatory) of Mushroom Crude Extracts ..................................... 24 2.3.1 Dose- and time-dependent MTT assay to determine the anti-proliferative activity of mushroom extracts on HeLa cell ........................................................................................................... 24 2.3.1.1 Dose-dependent MTT assay for assessment of growth-inhibitory activity of mushroom extracts ................................................................................................................................................ 24 2.3.2.2 Dose-dependent MTT assay protocol ..................................................................................... 25 2.3.2.3 Time-Dependent MTT assay for assessment of growth-inhibitory activity of mushroom extracts ................................................................................................................................................ 28 2.3.2 Assessing TNF- α production by RAW 264.7 cells as an indication of immuno-stimulation ...... 29 2.3.2.1 Enzyme-Linked Immunosorbent Assay (ELISA) ....................................................................... 29 2.3.3 Assessing inhibition of TNF-α production in RAW 264.7 cells as an indication of anti- inflammatory activity ............................................................................................................................. 30 2.4 Results and Discussion .......................................................................................................... 31 2.4.1 Genetic Identification of mushroom specimens 143 and 151. .................................................... 31 2.4.2 Chemical extraction of Canadian wild mushrooms ..................................................................... 33 2.4.3 Dose- and time-dependent anti-proliferative assessment of mushroom extracts using HeLa cells ......................................................................................................................................................... 35 2.4.3.1 Fomitopsis officinalis ............................................................................................................... 35 2.4.3.2 Trametes versicolor ................................................................................................................. 37 2.4.3.3 Cerrena unicolor ...................................................................................................................... 37 2.4.3.4 Tricholoma vaccinum .............................................................................................................. 38 2.4.3.5 Sarcodon scabripes ................................................................................................................. 39 2.4.3.6 Albatrellus flettii ...................................................................................................................... 41 2.4.4 Stimulation of TNF-alpha production in RAW 264.7 cells after treatment with mushroom extracts ................................................................................................................................................... 43 2.4.5 Inhibition of TNF-alpha production in LPS-induced RAW 264.7 cells .......................................... 46 2.5 Discussion............................................................................................................................... 50 2.6 Conclusion ............................................................................................................................. 53 Chapter 3: Purification, Identification and Characterization of Growth-inhibitory Compounds from Albatrellus flettii............................................................................................ 57 3.1 Introduction ..........................................................................................................................