Insights into the mechanism of safingol and its potential to synergize with anti cancer drugs

INSIGHTS INTO THE MECHANISM OF SAFINGOL AND ITS POTENTIAL TO SYNERGIZE WITH ANTICANCER DRUGS LING LEONG UUNG (B.Sc Pharm (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgement The PhD journey has been an uphill battle, just like the song “The Climb” There were countless moments fraught with hair-pulling frustration and disappointments Yet, these are the moments I will remember the most Nevertheless, this has been a rewarding journey because of the following people whom I would like to thank from the bottom of my heart Dr Gigi Chiu, my supervisor, whom I learnt first-hand lab skills from I would like to thank her for her constant support, encouragement and guidance during my studies It has been a really great pleasure to work under her supervision Thank you Dr Gigi! Thank you for sharing a lot of life experiences and stories with me You have taught and inspired me Of course, I would also like to thank Dr Pat for his assistance in flow cytometric analysis Bee Jen (the forever laboratory idol), who is always so kind and helpful Thank you for coaching me on doing experiments, especially for the tips you gave me on running Western blots (perhaps I still struggle ) And of course, I’m really glad to have travelled to Switzerland and Italy with you! My beloved laboratory mates, thank you very much! Man Yi, for always being a motivation to me and setting an example in the laboratory Anumita, thanks for all the sharing sessions and fun chats Shaikh, thanks for always reminding me that “it’s my last semester” each time we meet each other since my 3rd year Though somehow inducing “oxidative stress” to me, such a reminder did serve as a wake-up call (haha!) Kuan Boone, thanks for working with me on Safingol and discussing our grand plans for our experiments which we wanted to finish so quickly Hui Min, Rou Wei and Michelle, thanks for helping out with my experiments during their undergraduate studies Swee Eng, thanks for coordinating all the orders for laboratory supplies Without you, I think I would not have obtained all the reagents and chemicals on time to finish my thesis A/P Chan Sui Yung, the Head of Department, who has been very supportive and encouraging throughout these years I would like to thank you for giving me the opportunity to explore research for the first time during my undergraduate studies Doing research would never be the same without the company of graduate students Thanks to Hong May, Pay Chin, Ding Fung, Wen Qi, Wang Zhe, Zhan Yuin, Li Fang and Mandy (and the list goes on), who are willing to listen to my latest failed experiment or go for lunch/tea/dinner Thanks to the AAPSNUS Student Chapter and Pharmacy Graduate Society, where I built friendships with fellow graduate students! Not forgetting my dear friends in life- Jin San, Yin Yee, Sui Mui, Melissa, Shiau Fui, whom I have known for more than 15 years now Thank you all for i always being there for me and encouraging me through emails Thanks for all the fun and for speaking the same lingo as me when no one does Thanks Xn Yii, for your wonderful company and cooking all the delicious meal! Special thanks to Morgan, for standing by me all the time and lending me your listening ear even though you did not understand why I had to go back to the laboratory to “add drug” or “get result” at a specific time Thanks for being there and talking to me about everything under the sun, except research! My parents, for being so supportive and caring since I was born Thank you for calling me every week without fail I want to thank them for their patience, especially in waiting for me to graduate Last but not least, I want to thank God for everything, for His grace, mercy and love ii Table of Contents Acknowledgement .i Table of Contents iii Summary vi List of Tables ix List of Figures x List of Abbreviations xiii List of Publications xvii Chapter Introduction 1.1 Overview of Cancer 1.1.1 Conventional Treatment for Cancer 1.1.2 Rationale for Drug Combinations Approaches in Cancer 1.2 The Processes of Cell Death .8 1.2.1 Apoptosis 10 1.2.1.1 Intrinsic pathway 12 1.2.1.2 Extrinsic pathway 13 1.2.2 Necrosis 14 1.2.3 Autophagy .17 1.2.3.1 Autophagy and cell survival 21 1.2.3.2 Autophagy and cell death .21 1.2.4 Senescence 22 1.2.5 Mitotic catastrophe 23 1.3 Reactive Oxygen Species (ROS) 24 1.3.1 Biological sources and anti-oxidant defense mechanism of ROS 25 1.3.2 ROS and cancer 27 1.3.2.1 ROS and cell survival 28 1.3.2.2 ROS and cell death .29 1.4 Overview of sphingolipids 30 1.4.1 Sphingolipids and cell survival .32 1.4.2 Sphingolipids and cell death 34 1.4.3 Safingol 34 1.4.3.1 Molecular effects induced by safingol treatment 37 1.4.3.1.1 Protein kinase C (PKC) inhibition .38 1.4.3.1.2 Sphingosine kinase (SK) inhibition 41 1.4.3.1.3 Other molecular effects of safingol 45 1.4.3.2 Metabolism and toxicities 45 1.5 Thesis Rationale and Hypothesis 47 iii Chapter 2: Material and Methods 49 2.1 Materials 49 2.1.1 Reagents 49 2.1.2 Cell Lines 49 2.1.3 Antibodies .50 2.2 Cell viability assay 50 2.3 Flow Cytometry 52 2.3.1 Propidium iodide staining .52 2.3.2 Annexin V-FITC/7AAD staining 53 2.4 Protein analysis by Western blot .53 2.5 Cell adhesion assay 54 2.6 Electron microscopy 55 2.7 Mitochondria membrane potential measurement 56 2.8 ATP measurement 56 2.9 ROS detection assay 57 2.10 Acridine orange staining 58 2.11 Glucose uptake assay 58 2.12 Statistical analysis 59 Chapter 3: The role of protein kinase C in the synergistic interaction of safingol and irinotecan in colon cancer cells .60 3.1 Introduction .60 3.2 Results .62 3.2.1 Effect of safingol, irinotecan or 5-FU as single agents in colon cancer cells 62 3.2.2 Effect of fixed ratio combinations of safingol and irinotecan 65 3.2.3 Effect of safingol/irinotecan at 1:1 molar ratio on the cell cycle status of colon cancer cells .68 3.2.4 Role of PKC in mediating the cytotoxic effect of safingol and safingol/irinotecan combination .71 3.2.5 Effect of safingol/irinotecan at 1:1 molar ratio on colon cancer cell adhesion 75 3.3 Discussion .77 Chapter 4: The role of reactive oxygen species and autophagy in safingolinduced cell death 81 4.1 Introduction .81 4.2 Results .82 4.2.1 Safingol induced necrosis in MDA-MB-231 and HT-29 cells .82 4.2.2 Safingol induced ROS generation in MDA-MB-231 and HT-29 cancer cells 89 4.2.3 ROS trigger induction of autophagy in MDA-MB-231 and HT-29 cells 92 4.2.4 The role of autophagy and ROS in response to safingol treatment 97 iv 4.2.5 Bcl-xL and Bax regulates safingol-induced autophagy 99 4.2.6 Safingol reduced glucose uptake 102 4.3 Discussion 107 Chapter 5: Synergistic cytotoxic effect of safingol and conventional chemotherapeutic agents .112 5.1 Introduction 112 5.2 Results 115 5.2.1 Effect of safingol as a single agent in human cancer cell lines 115 5.2.2 Effect of fixed ratio combinations of safingol and conventional chemotherapeutic agents .115 5.2.3 Effect of fixed ratio combinations of safingol and conventional chemotherapeutic agents in the presence of NAC 121 5.3 Discussion .123 Chapter 6: Summarizing discussion 125 References 131 v Summary The increasing understanding of the cellular responses to anti-cancer drugs has revealed many new and effective opportunities for cancer therapy Safingol, which belongs to the family of sphingolipids, was originally developed as a protein kinase C (PKC) and sphingosine kinase (SK) inhibitor, and is currently evaluated in Phase I clinical trials Yet, the underlying mechanisms of its action remain largely unknown The research presented in this thesis focused on elucidating the mechanism of safingol and its potential to synergize with conventional anti-cancer drugs The results presented in Chapter demonstrated that, as a single agent, safingol was more potent than irinotecan and 5-fluorouracil (5-FU) in HT-29 and LS-174T colon cancer cell lines Furthermore, the combination of safingol/irinotecan at 1:1 molar ratio was found to be additive in HT-29 cells (CI = 0.94) and synergistic in LS-174T cells (CI = 0.68), and resulted in concentration- and time-dependent down-regulation of phosphorylated PKC and its downstream substrate, phosphorylated myristoylated alanine-rich Ckinase substrate (MARCKS) Observations that 1:1 safingol/irinotecan combination inhibited the adhesion of colon cancer cells to the extracellular matrix further supported the ability of this drug combination to modulate PKC downstream signaling These results suggested that modulation of the PKC pathway could be a possible molecular basis for the observed synergism of the safingol/irinotecan combination Intriguingly, the results showed that safingol as a single agent, however, did not inhibit PKC at the concentration that vi caused substantial cell kill This finding suggested that alternative molecular effects could be induced by safingol, and led to the study in Chapter The results summarized in Chapter are the first to document that safingol induced concentration- and time-dependent reactive oxygen species (ROS) generation in cancer cells, and ROS appeared to be a critical mediator in safingol-induced necrosis Depending on the levels of ROS generated, two opposite cellular responses were observed Low levels of ROS generation triggered autophagy which serves as a catabolic process to remove damaged organelles When the oxidative stress levels were high, cells died by necrosis In addition, the current results suggested that Bcl-xL and Bax are involved in the regulation of safingol-induced autophagy, despite their well-established roles in regulating apoptosis Furthermore, the results in Chapter suggested that safingol inhibited glucose uptake and activated AMP-activated protein kinase (AMPK) prior to ROS generation By elucidating the molecular effects of safingol, anti-cancer drug combinations based on safingol could be appropriately identified for effective treatment The aim of Chapter was to evaluate the activity of safingol in combination with various apoptotic- and ROS-generating chemotherapeutic agents using a variety of cancer cell lines Safingol was able to synergize with carboplatin, doxorubicin, gemcitabine and vincristine over a range of the fixed molar drug ratios tested In addition, the results in Chapter supported the notion that ROS was an important factor in mediating the observed synergism vii The use of safingol-based drug combinations holds therapeutic promise as an effective strategy for cancer therapy, and warrants future in vivo studies viii List of Tables Table 1.1 Trends in cancer incidence in Singapore 2003-2007……………….4 Table 1.2 Characteristic of different modes of cell death…………………… Table 3.1 IC50 values of safingol, irinotecan and 5-FU in HT-29 and LS-174T colon cancer cell lines ………………………………… 64 Table 3.2 Combination indices of safingol/irinotecan combinations administered in different molar ratios in colon cancer cell line… 67 Table 3.3 Percentage of HT-29 cells in various phases of the cell cycle after treated with safingol, irinotecan or safingol/irinotecan (1:1) for 24 h and 48 h ……………………………………………… 69 Table 3.4 Percentage of LS-174T cells in various phases of the cell cycle after treated with safingol, irinotecan or safingol/irinotecan (1:1) for 24 h and 48 h 70 Table 5.1 IC50 values of safingol and conventional anti-cancer drugs in respective cancer cell lines ……………………………………117 Table 5.2 Combination indices of safingol 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(OMM) This leads to the release of cytochrome c (Cyt c) from inner mitochondrial membrane space into the cytosol Another pro-apoptotic protein, second mitochondria-derived activator of caspase/direct... D) and the adenine nucleotide translocator (ANT), to enter into cytosol and induce cell death For O2·- which is released into the intermembrane space, they are dismuted to H2O2 by copper and