THE MECHANISTIC STUDIES OF THE ANTICANCER POTENTIAL OF ARTESUNATE IN HUMAN CANCER CELLS YANG NAIDI (M.Sc. Zhejiang University, P.R. China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2014 ACKNOWLEDGEMENTS I would like to express my most sincere and deepest gratitude to my supervisor, A/Prof. Shen Han-Ming for his professional and enthusiastic guidance throughout the past four years. This study would not have been possible without his excellent guidance, great supports and continuous encouragements. His enthusiasm and dedication to science have impressed and inspired me deeply. I have indeed gained fruitful experience for the ropes of biological research. What I have learned from him will not only benefit my future career but also my life. I would like to take this opportunity to delicate my sincere thanks to my thesis advisory committee members: A/Prof. Kevin, Tan Shyong Wei, and A/Prof. Reshma Taneja for their instructive suggestions and continuous supports on my study. I would also like to extend my gratefulness to the following people for providing materials for this study: Dr. N Mizushima (Tokyo Medical and Dental University, Japan) for providing the HeLa cells with stable expression of GFP-LC3; Dr. A Ballabio (Telethon Institute of Genetics and Medicine, Italy) for providing the TFEB-luciferase construct; Dr. DJ Kwiatkowski (Harvard University, USA) for providing the pair of Tsc2 WT and KO MEFs; Dr. Huang Jingxiang (National University Hospital, Singapore) for providing the pair of TSC2 WT and shTSC2 HeLa cells; Dr. TW Soong (National University of Singapore, Singapore) for providing Flag-FTH plasmid. iii It has been a great honor and fortune for me to work in such a warm and harmonious laboratory. I would like to specially thank Dr. Ng Shukie for her immense help in my study as well as daily life and also Dr. Tan Shi Hao for his helpful suggestions to my research. Special thanks also go to Mr. Ong Yeong Bing and Ms Su Jin for their logistical help. I would also like to express my deep appreciation to other lab members: Dr. Zhou Jing, Dr. Cui Jianzhou, Dr. Chen Bo, Ms Zhang Yin, Ms Shi Yin, Mr. Zhang Jianbin and Ms Mo Xiaofan for their supports and the friendship. Also, thank all other staffs in Saw Swee Hock School of Public Health and Department of Physiology, Yong Loo Lin School of Medicine. Especially, I would like to thank Dr. Tai Yee Kit (Department of Physiology, NUS) for the insightful discussions on my research project. Finally, I would like to extend my deep appreciation to my parents, younger sister for their endless love. Also numerous thanks to my husband Dr. Jiang Bo for his continuous love, support and understanding. iv PUBLICATIONS AND PRESENTATIONS AT CONFERENCES PUBLICATIONS 1. Yang ND, Tan SH, Ng S, Shi Y, Zhou J, Tan K SW, Wong WS F, Shen HM. (2014). Artesunate induces cell death in human cancer cells via enhancing lysosomal function and lysosomal degradation of ferritin. J Biol Chem 289, 33425-33441. 2. Zhang J, Ng S, Wang J, Tan SH, Zhou J, Yang ND, Lin Q, Xia D, Shen HM. (In press). Histone Deacetylase Inhibitors Induce Autophagy through FoxO1-Dependent Pathways. Autophagy. 3. Shi Y, Tan SH, Ng S, Yang ND, Zhou J, McMahon KA, Del Pozo MA, Hill MM, Parton RG, Kim YS, Shen HM. (In press). Caveolin-1 and lipid rafts in modulation of lysosomal function and autophagy in breast cancer cells. Autophagy. 4. Zhou J, Tan SH, Nicolas V, Bauvy C, Yang ND, Zhang J, Xue Y, Codogno P, and Shen HM. (2013). Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosomelysosome fusion. Cell Res 23, 508-523. 5. Zhang Y, Yang ND, Zhou F, Shen T, Duan T, Zhou J, Shi Y, Zhu XQ, and Shen HM. (2012). (-)-Epigallocatechin-3-gallate induces non-apoptotic cell death in human cancer cells via ROS-mediated lysosomal membrane permeabilization. PLoS One 7, e46749. v PRESENTATIONS AT CONFERENCES Yang ND, Tan SH, Ng S, Shi Y, Zhou J, Shen HM. Artesunate induces cancer cell death via enhancing the lysosomal degradation of ferritin. Gordon Research Conference, Autophagy in Stress, Development & Disease 16 – 21 Mar 2014, Renaissance Tuscany Il Ciocco Resort in Lucca (Barga) Italy. Yang ND, Tan SH, Ng S, Shi Y, Zhou J, Shen HM. Artesunate induces cancer cell death via enhancing the lysosomal degradation of ferritin. 7th Asia Pacific Organization of Cell Biology (APOCB) Congress & American Society for Cell Biology (ASCB) Workshops. 24 -27 Feb 2014, Biopolis, Singapore. Yang ND, Tan SH, Ng S, Shi Y, Zhou J, Shen HM. Artesunate induces cancer cell death via enhancing the lysosomal degradation of ferritin. International Conference on Natural Products and Health. 5-7 Sep 2013, Nanyang Technological University, Singapore. (Silver Best Poster Award) vi THE MECHANISTIC STUDIES OF THE ANTICANCER POTENTIAL OF ARTESUNATE IN HUMAN CANCER CELLS Table of Contents DECLARATION .ii ACKNOWLEDGEMENTS . iii PUBLICATION AND PRESENTATIONS AT CONFERENCES v PUBLICATIONS v PRESENTATIONS AT CONFERENCES vi SUMMARY xi LIST OF TABLES . xiii LIST OF FIGURES . xiii LIST OF ABBREVIATIONS . xvi CHAPTER 1. 1.1. INTRODUCTION . ARTESUNATE . 1.1.1 Overview of artemisinin and artesunate 1.1.2 Pharmacological effects of artesunate . 1.1.3 Molecular mechanisms underlying ART-mediated cell death in cancer cells . 14 1.2. AUTOPHAGY 19 1.2.1 Overview of autophagy 19 1.2.2 Stages of autophagy . 20 1.2.3 Biological functions of autophagy . 25 1.2.4 Implications of autophagy in human diseases . 29 1.3. REGULATION OF AUTOPHAGY BY MTORC1 AND LYSOSOMES . 34 vii 1.3.1 Regulation of autophagy by mTOR1 . 34 1.3.2 Regulation of autophagy by lysosomes . 37 1.3.3 Regulation of lysosomal function 38 1.4. IRON 42 1.4.1 Overview the role of iron in human body and cells . 42 1.4.2 Iron uptake regulated by TfR1 . 43 1.4.3 Iron storage protein ferritin 44 1.4.4 Iron responsive protein (IRP)/Iron responsive element (IRE) system 46 1.5. GAP OF KNOWLEDGE AND OBJECTIVES . 48 CHAPTER 2. MATERIAL AND METHODS . 50 2.1. CELL CULTURE . 51 2.2. CHEMICALS, REAGENTS, AND ANTIBODIES . 51 2.3. WESTERN BLOTTING 52 2.4. CONFOCAL IMAGING 53 2.5. CELL COLLECTION FOR FLOW CYTOMETRY 54 2.6. DETECTION OF CELL DEATH . 54 2.7. DETECTION OF THE INTRACELLULAR LOCALIZATION OF ART 54 2.8. LYSOTRACKER RED (LTR), LYSOTRACKER GREEN (LTG) AND MITOTRACKER RED (MTR) STAINING 55 2.9. MAGIC RED CATHEPSIN B AND L ACTIVITY ASSAY 55 2.10. DETERMINATION STAINING 56 2.11. IMMUNOFLUORESCENCE STAINING 56 OF PROTEIN PROTEOLYSIS USING viii DQ RED BSA 2.12. USE OF IN SITU PROXIMITY LIGATION ASSAY (PLA) ASSAY TO CHECK THE INTERACTION BETWEEN V1 AND V0 IN SITU . 57 2.13. SMALL INTERFERING RNA (SIRNA) AND TRANSIENT TRANSFECTION 57 2.14. MEASUREMENT OF ROS PRODUCTION . 58 2.15. LUCIFERASE ASSAYS 59 2.16. REVERSE TRANSCRIPTION AND QUANTITATIVE REAL-TIME PCR 60 2.17. STATISTICAL ANALYSIS 60 CHAPTER 3. ARTESUNATE INDUCES AUTOPHAGY AND ACTIVATES LYSOSOMAL FUNCTION . 61 3.1. INTRODUCTION 62 3.2. RESULTS 63 3.2.1 ART induces autophagy . 63 3.2.2 ART inhibits mTORC1 activity via the PI3K-Akt-TSC pathway 65 3.2.3 Accumulation of ART in the lysosomes is independent of lysosomal pH . 69 3.2.4 Artesunate activates lysosomal function 73 3.2.5 ART treatment does not increase lysosomal number 79 3.2.6 Mechanisms of lysosomal activation by ART . 81 CHAPTER 4. FERRITIN DEGRADATION IS REQUIRED FOR ART-INDUCED CANCER CELL DEATH . 89 4.1. INTRODUCTION 90 4.2. RESULTS 92 ix 4.2.1 ART inhibits cell proliferation and induces cell death in human cancer cells . 92 4.2.2 ART induces apoptotic cell death in human cancer cells 95 4.2.3 ART induces oxidative stress . 98 4.2.4 Lysosomes functions as the upstream of mitochondrial ROS production 100 4.2.5 Lysosomal activation, ROS production and cell death induced by ART is dependent on lysosomal iron 104 4.2.6 ART promotes ferritin degradation in the lysosomes 107 4.2.7 Overexpression of FTH reduces ART-induced cell death . 112 4.2.8 Autophagy plays a marginal role in ART-induced cell death 114 4.2.9 Lysosomal delivery and degradation of ferritin is required for ART-induced cell death . 116 CHAPTER 5. GENERAL DISCUSSION AND CONCLUSIONS 118 5.1. THE EFFECT OF ART ON AUTOPHAGY 119 5.2. THE EFFECT OF ART ON LYSOSOMES . 121 5.3. THE IMPORTANCE OF ROS IN ART-INDUCED CELL DEATH 122 5.4. THE ROLE OF IRON IN ART-INDUCED LYSOSOMAL ACTIVATION 123 5.5. 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Nat Rev Mol Cell Biol 12, 21-35. 154 [...]... of malaria (Sinclair et al., 2011) There are also several other derivatives of artemisinin including artemether, arteether and dihydroartemisinin (DHA) are also wildly used as anti-malaria drugs The endoperoxide bridges of artemisinins are believed to be responsible for the mechanism of action The successful identification of artemisinin and development of ART as the first-line drug for treatment of. .. responses of eosinophils, T and B lymphocytes, and mast cells, Cheng et al investigated the antiinflammatory effect of ART in ovalbumin (OVA)-induced inflammatory mice as well as in house dust mite induced mouse asthma model (Cheng et al., 2011) They found that ART inhibited the OVA-induced phosphorylation of Akt They further made use of primary human bronchial epithelial cells and found that EGF-induced... ovalbumin phagophore assembly site phosphate buffer saline phosphoinositide-dependent kinase phosphatidylethanolamine paraformaldehyde phosphoinositide 3- kinase protein kinase B protein kinase C proximity ligation assay quantitative real-time PCR regulatory-associated protein of mTOR rapamycin-insensitive companion of mTOR reactive oxygen species Beclin-1 interacting and cystein-rich containing S6 kinase... one clinical trial ongoing using ART in metastatic breast cancer (http://clinicaltrials.gov/ct2/show/NCT00764036) and the results are still pending Figure 1.2 Anti -cancer effects of ART 8 (i) Arrest of cell cycle and inhibition of cell proliferation One hallmark of cancer is its sustaining proliferative signaling (Hanahan and Weinberg, 2011) There are a number of studies showing the cell cycle arrest... of the endoperoxide bridge of artemisinins by ferrous iron results in carbon-centered free radicals production, which is essential for their anti-malarial activity (Eckstein-Ludwig et al., 2003; Klonis et al., 2013) The underlying mechanisms of the anti-malaria function of artemisinins have been extensively studied, including: (i) inhibition the PfATP6 of Plasmodium falciparum in Xenopus oocytes, the. .. endothelial growth factor xvii CHAPTER 1 INTRODUCTION 1 1.1 ARTESUNATE 1.1.1 Overview of artemisinin and artesunate Artemisinin, an active ingredient of a traditional Chinese medicinal plant Artemisia annua L (qinhao), has been widely used for treatment of fever and chills caused by malaria infections (Klayman, 1985) Artesunate (ART), a water soluble derivate of artemisinin, was found to be one of the. .. 2008), antiinflammatory (Wang et al., 2007; Xu et al., 2007), anti-allergic and asthmatic activities (Cheng et al., 2011) There are continuous efforts and increasing interests in uncovering the underlying mechanisms of the above functions 1.1.2.1 Anti-malaria As shown in Figure 1.1, the basic structure of artemisinin and its monomers including ART is a sesquiterpene lactone All of them contain an 3 endoperoxide... is the only mechanism that involves in 9 executing the cell death without any signature of apoptosis or necrosis, which will be discussed in detail later (Scarlatti et al., 2009) Generally, there are two main pathways initiating apoptosis: the extrinsic death pathway which initiates by binding of death receptor ligands to specific death receptors on the cell surface and the intrinsic pathway which initiates... suggesting the anti -cancer function of artemisinins, especially ART and DHA (Ho et al., 2014) Here we focus on the anti -cancer function of ART Up to date, the anti -cancer function of ART is mainly based on the following observations: (i) induction of cell cycle arrest (Longxi et al., 2011; Zhao et al., 2011), (ii) induction of cell death and sensitization to tumor necrosis factorrelated apoptosis-inducing... Gaynor, 2001) Therefore, inactivation of NF-κB leads to the repression of production of key proinflammatory cytokines of such as TNF-α, IL-1, IL-6, IL-12, reduction of the expression of enzymes such as nitric oxide synthase and inhibition of the activation of immunocompetent cells (Lawrence et al., 2001) It has been suggested that ART is capable of suppressing TNF-α induced production of IL-1, IL-6 . THE MECHANISTIC STUDIES OF THE ANTICANCER POTENTIAL OF ARTESUNATE IN HUMAN CANCER CELLS YANG NAIDI (M.Sc. Zhejiang University, P.R. China) A THESIS SUBMITTED FOR THE DEGREE OF. Singapore. (Silver Best Poster Award) vii THE MECHANISTIC STUDIES OF THE ANTICANCER POTENTIAL OF ARTESUNATE IN HUMAN CANCER CELLS Table of Contents DECLARATION ii ACKNOWLEDGEMENTS iii PUBLICATION. ART induced autophagy in human cervical cancer HeLa cells evidenced by the increase of autophagic flux. In the search of the mechanisms for ART-induced autophagy, we found that ART inhibits mechanistic/ mammalian