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 with different classes of anti-cancer drugs in respective cancer cell lines ……………… 119 ix 28 Hunter DR, Haworth RA The Ca2+-induced membrane transition in mitochondria I The protective mechanisms Arch Biochem Biophys 1979 Jul; 195(2): 453-459 29 Bano D, Young KW, Guerin CJ, Lefeuvre R, Rothwell NJ, Naldini L, et al Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitotoxicity Cell 2005 Jan 28; 120(2): 275-285 30 Festjens N, Vanden Berghe T, Cornelis S, Vandenabeele P RIP1, a kinase on the crossroads of a cell's decision to live or die Cell Death Differ 2007 Mar; 14(3): 400-410 31 Vercammen D, Vandenabeele P, Beyaert R, Declercq W, Fiers W Tumour necrosis factor-induced necrosis versus anti-Fas-induced apoptosis in L929 cells Cytokine 1997 Nov; 9(11): 801-808 32 Ma Y, Temkin V, Liu H, Pope RM NF-kappaB protects macrophages from lipopolysaccharide-induced cell death: the role of caspase and receptor-interacting protein J Biol Chem 2005 Dec 23; 280(51): 41827-41834 33 Eliasson MJ, Sampei K, Mandir AS, Hurn PD, Traystman RJ, Bao J, et al Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia Nat Med 1997 Oct; 3(10): 1089-1095 34 Xu Y, Huang S, Liu ZG, Han J Poly(ADP-ribose) polymerase-1 signaling to mitochondria in necrotic cell death requires RIP1/TRAF2mediated JNK1 activation J Biol Chem 2006 Mar 31; 281(13): 87888795 35 Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, et al Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule Nat Immunol 2000 Dec; 1(6): 489-495 36 Harper N, Hughes M, MacFarlane M, Cohen GM Fas-associated death domain protein and caspase-8 are not recruited to the tumor necrosis factor receptor signaling complex during tumor necrosis factorinduced apoptosis J Biol Chem 2003 Jul 11; 278(28): 25534-25541 37 Klionsky DJ Autophagy: from phenomenology to molecular understanding in less than a decade Nat Rev Mol Cell Biol 2007 Nov; 8(11): 931-937 38 Levine B, Kroemer G Autophagy in aging, disease and death: the true identity of a cell death impostor Cell Death Differ 2009 Jan; 16(1): 12 39 Klionsky DJ Autophagy Curr Biol 2005 Apr 26; 15(8): R282-283 133 40 Kondo Y, Kanzawa T, Sawaya R, Kondo S The role of autophagy in cancer development and response to therapy Nat Rev Cancer 2005 2005 Sep; 5(9): 726-734 41 Levine B, Klionsky DJ Development by self-digestion: molecular mechanisms and biological functions of autophagy Dev Cell 2004 Apr; 6(4): 463-477 42 Mizushima N [Methods for monitoring macroautophagy] Tanpakushitsu Kakusan Koso 2006 2006 Aug; 51(10 Suppl): 15421548 43 Mariño G, López-Otín C Autophagy: molecular mechanisms, physiological functions and relevance in human pathology Cell Mol Life Sci 2004 2004 Jun; 61(12): 1439-1454 44 Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, et al LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing EMBO J 2000 Nov 1; 19(21): 5720-5728 45 Liang XH, Kleeman LK, Jiang HH, Gordon G, Goldman JE, Berry G, et al Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein J Virol 1998 Nov; 72(11): 8586-8596 46 Hanada T, Noda NN, Satomi Y, Ichimura Y, Fujioka Y, Takao T, et al The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy J Biol Chem 2007 Dec 28; 282(52): 3729837302 47 Maiuri M, Zalckvar E, Kimchi A, Kroemer G Self-eating and selfkilling: crosstalk between autophagy and apoptosis Nat Rev Mol Cell Biol 2007 2007 Sep; 8(9): 741-752 48 Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, et al Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy Cell 2005 Sep 23; 122(6): 927-939 49 White E Autophagic cell death unraveled: Pharmacological inhibition of apoptosis and autophagy enables necrosis Autophagy 2008 May 16; 4(4): 399-401 50 Galluzzi L, Vicencio JM, Kepp O, Tasdemir E, Maiuri MC, Kroemer G To die or not to die: that is the autophagic question Curr Mol Med 2008 Mar; 8(2): 78-91 51 Toth S, Nagy K, Palfia Z, Rez G Cellular autophagic capacity changes during azaserine-induced tumour progression in the rat pancreas Upregulation in all premalignant stages and down-regulation with loss of 134 cycloheximide sensitivity of segregation along with malignant transformation Cell Tissue Res 2002 Sep; 309(3): 409-416 52 Boya P, Gonzalez-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, et al Inhibition of macroautophagy triggers apoptosis Mol Cell Biol 2005 Feb; 25(3): 1025-1040 53 Shimizu S, Kanaseki T, Mizushima N, Mizuta T, Arakawa-Kobayashi S, Thompson CB, et al Role of Bcl-2 family proteins in a nonapoptotic programmed cell death dependent on autophagy genes Nat Cell Biol 2004 Dec; 6(12): 1221-1228 54 Bursch W, Ellinger A, Kienzl H, Torok L, Pandey S, Sikorska M, et al Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy Carcinogenesis 1996 Aug; 17(8): 1595-1607 55 Kanzawa T, Kondo Y, Ito H, Kondo S, Germano I Induction of autophagic cell death in malignant glioma cells by arsenic trioxide Cancer Res 2003 2003 May; 63(9): 2103-2108 56 Singh R, George J, Shukla Y Role of senescence and mitotic catastrophe in cancer therapy Cell Div 2010; 5: 57 Saretzki G Cellular senescence in the development and treatment of cancer Curr Pharm Des 2010 Jan; 16(1): 79-100 58 Shay JW, Pereira-Smith OM, Wright WE A role for both RB and p53 in the regulation of human cellular senescence Exp Cell Res 1991 Sep; 196(1): 33-39 59 Castedo M, Perfettini JL, Roumier T, Andreau K, Medema R, Kroemer G Cell death by mitotic catastrophe: a molecular definition Oncogene 2004 Apr 12; 23(16): 2825-2837 60 Vakifahmetoglu H, Olsson M, Zhivotovsky B Death through a tragedy: mitotic catastrophe Cell Death Differ 2008 Jul; 15(7): 1153-1162 61 Roninson IB, Broude EV, Chang BD If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells Drug Resist Updat 2001 Oct; 4(5): 303-313 62 Ianzini F, Bertoldo A, Kosmacek EA, Phillips SL, Mackey MA Lack of p53 function promotes radiation-induced mitotic catastrophe in mouse embryonic fibroblast cells Cancer Cell Int 2006; 6: 11 63 Azad MB, Chen Y, Gibson SB Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and treatment Antioxid Redox Signal 2009 Apr; 11(4): 777-790 135 64 Lau AT, Wang Y, Chiu JF Reactive oxygen species: current knowledge and applications in cancer research and therapeutic J Cell Biochem 2008 May 15; 104(2): 657-667 65 Finkel T, Holbrook NJ Oxidants, oxidative stress and the biology of ageing Nature 2000 Nov 9; 408(6809): 239-247 66 Boveris A, Oshino N, Chance B The cellular production of hydrogen peroxide Biochem J 1972 Jul; 128(3): 617-630 67 Cadenas E, Boveris A, Ragan CI, Stoppani AO Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria Arch Biochem Biophys 1977 Apr 30; 180(2): 248-257 68 Loschen G, Azzi A, Flohe L Mitochondrial H2O2 formation: relationship with energy conservation FEBS Lett 1973 Jun 15; 33(1): 84-87 69 Poyton RO, Ball KA, Castello PR Mitochondrial generation of free radicals and hypoxic signaling Trends Endocrinol Metab 2009 Sep; 20(7): 332-340 70 Mannella CA, Forte M, Colombini M Toward the molecular structure of the mitochondrial channel, VDAC J Bioenerg Biomembr 1992 Feb; 24(1): 7-19 71 Kuan J, Saier MH, Jr The mitochondrial carrier family of transport proteins: structural, functional, and evolutionary relationships Crit Rev Biochem Mol Biol 1993; 28(3): 209-233 72 Toyokuni S, Okamoto K, Yodoi J, Hiai H Persistent oxidative stress in cancer FEBS Lett 1995 Jan 16; 358(1): 1-3 73 Szatrowski TP, Nathan CF Production of large amounts of hydrogen peroxide by human tumor cells Cancer Res 1991 Feb 1; 51(3): 794798 74 Zhou Y, Hileman EO, Plunkett W, Keating MJ, Huang P Free radical stress in chronic lymphocytic leukemia cells and its role in cellular sensitivity to ROS-generating anticancer agents Blood 2003 May 15; 101(10): 4098-4104 75 Patel BP, Rawal UM, Dave TK, Rawal RM, Shukla SN, Shah PM, et al Lipid peroxidation, total antioxidant status, and total thiol levels predict overall survival in patients with oral squamous cell carcinoma Integr Cancer Ther 2007 Dec; 6(4): 365-372 136 76 Tsao SM, Yin MC, Liu WH Oxidant stress and B vitamins status in patients with non-small cell lung cancer Nutr Cancer 2007; 59(1): 813 77 Janssen AM, Bosman CB, Kruidenier L, Griffioen G, Lamers CB, van Krieken JH, et al Superoxide dismutases in the human colorectal cancer sequence J Cancer Res Clin Oncol 1999; 125(6): 327-335 78 Kanbagli O, Ozdemirler G, Bulut T, Yamaner S, Aykac-Toker G, Uysal M Mitochondrial lipid peroxides and antioxidant enzymes in colorectal adenocarcinoma tissues Jpn J Cancer Res 2000 Dec; 91(12): 1258-1263 79 Nicotera TM, Privalle C, Wang TC, Oshimura M, Barrett JC Differential proliferative responses of Syrian hamster embryo fibroblasts to paraquat-generated superoxide radicals depending on tumor suppressor gene function Cancer Res 1994 Jul 15; 54(14): 3884-3888 80 Murrell GA, Francis MJ, Bromley L Modulation of fibroblast proliferation by oxygen free radicals Biochem J 1990 Feb 1; 265(3): 659-665 81 Kaltschmidt B, Sparna T, Kaltschmidt C Activation of NF-kappa B by reactive oxygen intermediates in the nervous system Antioxid Redox Signal 1999 Summer; 1(2): 129-144 82 Pouyssegur J, Dayan F, Mazure NM Hypoxia signalling in cancer and approaches to enforce tumour regression Nature 2006 May 25; 441(7092): 437-443 83 Gopalakrishna R, Jaken S Protein kinase C signaling and oxidative stress Free Radic Biol Med 2000 May 1; 28(9): 1349-1361 84 Scherz-Shouval R, Elazar Z ROS, mitochondria and the regulation of autophagy Trends Cell Biol 2007 Sep; 17(9): 422-427 85 Chen Y, Azad MB, Gibson SB Superoxide is the major reactive oxygen species regulating autophagy Cell Death Differ 2009 Jul; 16(7): 1040-1052 86 Huang Q, Wu YT, Tan HL, Ong CN, Shen HM A novel function of poly(ADP-ribose) polymerase-1 in modulation of autophagy and necrosis under oxidative stress Cell Death Differ 2009 Feb; 16(2): 264-277 87 Kim EH, Sohn S, Kwon HJ, Kim SU, Kim MJ, Lee SJ, et al Sodium selenite induces superoxide-mediated mitochondrial damage and subsequent autophagic cell death in malignant glioma cells Cancer Res 2007 Jul 1; 67(13): 6314-6324 137 88 Kim EH, Choi KS A critical role of superoxide anion in seleniteinduced mitophagic cell death Autophagy 2008 Jan 1; 4(1): 76-78 89 Fleury C, Mignotte B, Vayssiere JL Mitochondrial reactive oxygen species in cell death signaling Biochimie 2002 Feb-Mar; 84(2-3): 131141 90 Stadtman ER Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences Free Radic Biol Med 1990; 9(4): 315-325 91 Simon HU, Haj-Yehia A, Levi-Schaffer F Role of reactive oxygen species (ROS) in apoptosis induction Apoptosis 2000 Nov; 5(5): 415418 92 Higuchi Y Chromosomal DNA fragmentation in apoptosis and necrosis induced by oxidative stress Biochem Pharmacol 2003 Oct 15; 66(8): 1527-1535 93 Dipple A DNA adducts of chemical carcinogens Carcinogenesis 1995 Mar; 16(3): 437-441 94 Chen Q, Ames BN Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells Proc Natl Acad Sci U S A 1994 May 10; 91(10): 4130-4134 95 Ogretmen B, Hannun YA Biologically active sphingolipids in cancer pathogenesis and treatment Nat Rev Cancer 2004 Aug; 4(8): 604-616 96 Futerman AH, Hannun YA The complex life of simple sphingolipids EMBO Rep 2004 Aug; 5(8): 777-782 97 Hannun YA, Obeid LM Principles of bioactive lipid signalling: lessons from sphingolipids Nat Rev Mol Cell Biol 2008 Feb; 9(2): 139-150 98 Obeid LM, Linardic CM, Karolak LA, Hannun YA Programmed cell death induced by ceramide Science 1993 Mar 19; 259(5102): 17691771 99 Hla T Physiological and pathological actions of sphingosine 1phosphate Semin Cell Dev Biol 2004 Oct; 15(5): 513-520 100 Spiegel S, Milstien S Sphingosine-1-phosphate: signaling inside and out FEBS Lett 2000 Jun 30; 476(1-2): 55-57 101 Cuvillier O, Levade T Sphingosine 1-phosphate antagonizes apoptosis of human leukemia cells by inhibiting release of cytochrome c and 138 Smac/DIABLO from mitochondria Blood 2001 Nov 1; 98(9): 28282836 102 Lee OH, Lee DJ, Kim YM, Kim YS, Kwon HJ, Kim KW, et al Sphingosine 1-phosphate stimulates tyrosine phosphorylation of focal adhesion kinase and chemotactic motility of endothelial cells via the G(i) protein-linked phospholipase C pathway Biochem Biophys Res Commun 2000 Feb 5; 268(1): 47-53 103 Nava VE, Hobson JP, Murthy S, Milstien S, Spiegel S Sphingosine kinase type promotes estrogen-dependent tumorigenesis of breast cancer MCF-7 cells Exp Cell Res 2002 Nov 15; 281(1): 115-127 104 Johnson KR, Johnson KY, Crellin HG, Ogretmen B, Boylan AM, Harley RA, et al Immunohistochemical distribution of sphingosine kinase in normal and tumor lung tissue J Histochem Cytochem 2005 Sep; 53(9): 1159-1166 105 Kawamori T, Osta W, Johnson KR, Pettus BJ, Bielawski J, Tanaka T, et al Sphingosine kinase is up-regulated in colon carcinogenesis FASEB J 2006 Feb; 20(2): 386-388 106 Takabe K, Paugh SW, Milstien S, Spiegel S "Inside-out" signaling of sphingosine-1-phosphate: therapeutic targets Pharmacol Rev 2008 Jun; 60(2): 181-195 107 Ogretmen B Sphingolipids in cancer: regulation of pathogenesis and therapy FEBS Lett 2006 Oct 9; 580(23): 5467-5476 108 Dragusin M, Gurgui C, Schwarzmann G, Hoernschemeyer J, van Echten-Deckert G Metabolism of the unnatural anticancer lipid safingol, L-threo-dihydrosphingosine, in cultured cells J Lipid Res 2003 2003 Sep; 44(9): 1772-1779 109 Schwartz G, Jiang J, Kelsen D, Albino A Protein kinase C: a novel target for inhibiting gastric cancer cell invasion J Natl Cancer Inst 1993 1993 Mar; 85(5): 402-407 110 Stoffel W, Hellenbroich B, Heimann G Properties and specificities of sphingosine kinase from blood platelets Hoppe Seylers Z Physiol Chem 1973 Oct-Nov; 354(10-11): 1311-1316 111 Rajewski R, Kosednar D, Matches T, Wong O, Burchett K, Thakker K Stereo-specific analysis of a novel protein kinase C inhibitor J Pharm Biomed Anal 1995 1995 Mar; 13(3): 247-253 112 Buehrer BM, Bell RM Inhibition of sphingosine kinase in vitro and in platelets Implications for signal transduction pathways J Biol Chem 1992 Feb 15; 267(5): 3154-3159 139 113 Avanti PL Product Data Sheet 114 Schwartz G, Ward D, Saltz L, Casper E, Spiess T, Mullen E, et al A pilot clinical/pharmacological study of the protein kinase C-specific inhibitor safingol alone and in combination with doxorubicin Clin Cancer Res 1997 1997 Apr; 3(4): 537-543 115 Carvajal RD A phase I clinical trial of safingol followed by cisplatin: Promising activity in refractory adrenocortical cancer with novel pharmacology Proc Amer Soc Clin Oncol 2006; 24: 13044 116 Schwartz G, Haimovitz-Friedman A, Dhupar S, Ehleiter D, Maslak P, Lai L, et al Potentiation of apoptosis by treatment with the protein kinase C-specific inhibitor safingol in mitomycin C-treated gastric cancer cells J Natl Cancer Inst 1995 1995 Sep; 87(18): 1394-1399 117 Hoffmann T, Leenen K, Hafner D, Balz V, Gerharz C, Grund A, et al Antitumor activity of protein kinase C inhibitors and cisplatin in human head and neck squamous cell carcinoma lines Anticancer Drugs 2002 2002 Jan; 13(1): 93-100 118 Jarvis W, Fornari FJ, Tombes R, Erukulla R, Bittman R, Schwartz G, et al Evidence for involvement of mitogen-activated protein kinase, rather than stress-activated protein kinase, in potentiation of 1-beta-Darabinofuranosylcytosine-induced apoptosis by interruption of protein kinase C signaling Mol Pharmacol 1998 1998 Nov; 54(5): 844-856 119 Maurer B, Melton L, Billups C, Cabot M, Reynolds C Synergistic cytotoxicity in solid tumor cell lines between N-(4hydroxyphenyl)retinamide and modulators of ceramide metabolism J Natl Cancer Inst 2000 2000 Dec; 92(23): 1897-1909 120 Sachs C, Safa A, Harrison S, Fine R Partial inhibition of multidrug resistance by safingol is independent of modulation of P-glycoprotein substrate activities and correlated with inhibition of protein kinase C J Biol Chem 1995 1995 Nov; 270(44): 26639-26648 121 Hannun Y, Loomis C, Bell R Protein kinase C activation in mixed micelles Mechanistic implications of phospholipid, diacylglycerol, and calcium interdependencies J Biol Chem 1986 1986 Jun; 261(16): 7184-7190 122 Kedderis L, Bozigian H, Kleeman J, Hall R, Palmer T, Harrison SJ, et al Toxicity of the protein kinase C inhibitor safingol administered alone and in combination with chemotherapeutic agents Fundam Appl Toxicol 1995 1995 May; 25(2): 201-217 123 Sliva D, English D, Lyons D, Lloyd FP, Jr Protein kinase C induces motility of breast cancers by upregulating secretion of urokinase-type 140 plasminogen activator through activation of AP-1 and NF-kappaB Biochem Biophys Res Commun 2002 Jan 11; 290(1): 552-557 124 Newton AC Regulation of protein kinase C Curr Opin Cell Biol 1997 Apr; 9(2): 161-167 125 Ron D, Kazanietz MG New insights into the regulation of protein kinase C and novel phorbol ester receptors FASEB J 1999 Oct; 13(13): 1658-1676 126 Hamada M, Sumi T, Iwai S, Nakazawa M, Yura Y Induction of endonuclease G-mediated apopotosis in human oral squamous cell carcinoma cells by protein kinase C inhibitor safingol Apoptosis 2006 2006 Jan; 11(1): 47-56 127 Coward J, Ambrosini G, Musi E, Truman JP, Haimovitz-Friedman A, Allegood JC, et al Safingol (L-threo-sphinganine) induces autophagy in solid tumor cells through inhibition of PKC and the PI3-kinase pathway Autophagy 2009 Feb; 5(2): 184-193 128 Hannun Y, Loomis C, Merrill AJ, Bell R Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets J Biol Chem 1986 1986 Sep; 261(27): 1260412609 129 Leclercq TM, Pitson SM Cellular signalling by sphingosine kinase and sphingosine 1-phosphate IUBMB Life 2006 Aug; 58(8): 467-472 130 Buehrer B, Bell R Sphingosine kinase: properties and cellular functions Adv Lipid Res 1993; 26: 59-67 131 Zeidan Y, Hannun Y Translational aspects of sphingolipid metabolism Trends Mol Med 2007 2007 Aug; 13(8): 327-336 132 Xia P, Gamble JR, Wang L, Pitson SM, Moretti PA, Wattenberg BW, et al An oncogenic role of sphingosine kinase Curr Biol 2000 Nov 30; 10(23): 1527-1530 133 French KJ, Schrecengost RS, Lee BD, Zhuang Y, Smith SN, Eberly JL, et al Discovery and evaluation of inhibitors of human sphingosine kinase Cancer Res 2003 Sep 15; 63(18): 5962-5969 134 Olivera A, Spiegel S Sphingosine-1-phosphate as second messenger in cell proliferation induced by PDGF and FCS mitogens Nature 1993 Oct 7; 365(6446): 557-560 135 Pitson SM, Xia P, Leclercq TM, Moretti PA, Zebol JR, Lynn HE, et al Phosphorylation-dependent translocation of sphingosine kinase to the plasma membrane drives its oncogenic signalling J Exp Med 2005 Jan 3; 201(1): 49-54 141 136 Taha TA, Hannun YA, Obeid LM Sphingosine kinase: biochemical and cellular regulation and role in disease J Biochem Mol Biol 2006 Mar 31; 39(2): 113-131 137 Melendez AJ, Carlos-Dias E, Gosink M, Allen JM, Takacs L Human sphingosine kinase: molecular cloning, functional characterization and tissue distribution Gene 2000 Jun 13; 251(1): 19-26 138 Liu H, Sugiura M, Nava VE, Edsall LC, Kono K, Poulton S, et al Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type isoform J Biol Chem 2000 Jun 30; 275(26): 19513-19520 139 Yokota S, Taniguchi Y, Kihara A, Mitsutake S, Igarashi Y Asp177 in C4 domain of mouse sphingosine kinase 1a is important for the sphingosine recognition FEBS Lett 2004 Dec 3; 578(1-2): 106-110 140 Morales P, Dillehay D, Moody S, Pallas D, Pruett S, Allgood J, et al Safingol toxicology after oral administration to TRAMP mice: demonstration of safingol uptake and metabolism by N-acylation and N-methylation Drug Chem Toxicol 2007; 30(3): 197-216 141 Noda T, Iwai S, Hamada M, Fujita Y, Yura Y Induction of apoptosis of detached oral squamous cell carcinoma cells by safingol Possible role of Bim, focal adhesion kinase and endonuclease G Apoptosis 2009 Mar; 14(3): 287-297 142 Venkataraman K, Futerman AH Comparison of the metabolism of Lerythro- and L-threo-sphinganines and ceramides in cultured cells and in subcellular fractions Biochim Biophys Acta 2001 Feb 26; 1530(2-3): 219-226 143 Schmitt CA, Lowe SW Apoptosis is critical for drug response in vivo Drug Resist Updat 2001 Apr; 4(2): 132-134 144 Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ Cancer statistics, 2009 CA Cancer J Clin 2009 Jun 145 Rothenberg ML Topoisomerase I inhibitors: review and update Ann Oncol 1997 Sep; 8(9): 837-855 146 Pizzolato JF, Saltz LB The camptothecins Lancet 2003 Jun 28; 361(9376): 2235-2242 147 Fuchs C, Mitchell EP, Hoff PM Irinotecan in the treatment of colorectal cancer Cancer Treat Rev 2006 Nov; 32(7): 491-503 148 Zhang N, Yin Y, Xu S, Chen W 5-Fluorouracil: mechanisms of resistance and reversal strategies Molecules 2008; 13(8): 1551-1569 142 149 Litvak D, Bilchik A, Cabot M Modulators of ceramide metabolism sensitize colorectal cancer cells to chemotherapy: a novel treatment strategy J Gastrointest Surg 2003 Jan; 7(1): 140-148; discussion 148 150 Van Cutsem E, Blijham GH Irinotecan versus infusional 5fluorouracil: a phase III study in metastatic colorectal cancer following failure on first-line 5-fluorouracil V302 Study Group Semin Oncol 1999 Feb; 26(1 Suppl 5): 13-20 151 Schwartz G, Farsi K, Maslak P, Kelsen D, Spriggs D Potentiation of apoptosis by flavopiridol in mitomycin-C-treated gastric and breast cancer cells Clin Cancer Res 1997 1997 Sep; 3(9): 1467-1472 152 Masur K, Lang K, Niggemann B, Zanker KS, Entschladen F High PKC alpha and low E-cadherin expression contribute to high migratory activity of colon carcinoma cells Mol Biol Cell 2001 Jul; 12(7): 19731982 153 O'Brian CA, Ward NE, Gravitt KR, Gupta KP The tumor promoter receptor protein kinase C: a novel target for chemoprevention and therapy of human colon cancer Prog Clin Biol Res 1995; 391: 117120 154 Jimeno A, Rudek M, Purcell T, Laheru D, Messersmith W, Dancey J, et al Phase I and pharmacokinetic study of UCN-01 in combination with irinotecan in patients with solid tumors Cancer Chemother Pharmacol 2008 Mar; 61(3): 423-433 155 Jones C, Clements M, Wasi S, Daoud S Enhancement of camptothecin-induced cytotoxicity with UCN-01 in breast cancer cells: abrogation of S/G(2) arrest Cancer Chemother Pharmacol 2000; 45(3): 252-258 156 Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters J Biol Chem 1982 1982 Jul; 257(13): 7847-7851 157 Combadiere C, Pedruzzi E, Hakim J, Perianin A A protein kinase inhibitor, staurosporine, enhances the expression of phorbol dibutyrate binding sites in human polymorphonuclear leucocytes Biochem J 1993 Feb 1; 289 ( Pt 3): 695-701 158 Ways DK, Kukoly CA, deVente J, Hooker JL, Bryant WO, Posekany KJ, et al MCF-7 breast cancer cells transfected with protein kinase Calpha exhibit altered expression of other protein kinase C isoforms and display a more aggressive neoplastic phenotype J Clin Invest 1995 Apr; 95(4): 1906-1915 143 159 Villalona-Calero MA, Ritch P, Figueroa JA, Otterson GA, Belt R, Dow E, et al A phase I/II study of LY900003, an antisense inhibitor of protein kinase C-alpha, in combination with cisplatin and gemcitabine in patients with advanced non-small cell lung cancer Clin Cancer Res 2004 Sep 15; 10(18 Pt 1): 6086-6093 160 Grossman SA, Alavi JB, Supko JG, Carson KA, Priet R, Dorr FA, et al Efficacy and toxicity of the antisense oligonucleotide aprinocarsen directed against protein kinase C-alpha delivered as a 21-day continuous intravenous infusion in patients with recurrent high-grade astrocytomas Neuro Oncol 2005 Jan; 7(1): 32-40 161 Yuen AR, Halsey J, Fisher GA, Holmlund JT, Geary RS, Kwoh TJ, et al Phase I study of an antisense oligonucleotide to protein kinase Calpha (ISIS 3521/CGP 64128A) in patients with cancer Clin Cancer Res 1999 Nov; 5(11): 3357-3363 162 Schmitt M, Harbeck N, Thomssen C, Wilhelm O, Magdolen V, Reuning U, et al Clinical impact of the plasminogen activation system in tumor invasion and metastasis: prognostic relevance and target for therapy Thromb Haemost 1997 Jul; 78(1): 285-296 163 Cheng J, Park TS, Chio LC, Fischl AS, Ye XS Induction of apoptosis by sphingoid long-chain bases in Aspergillus nidulans Mol Cell Biol 2003 Jan; 23(1): 163-177 164 Shi L, Bielawski J, Mu J, Dong H, Teng C, Zhang J, et al Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis Cell Res 2007 Dec; 17(12): 1030-1040 165 Villena J, Henriquez M, Torres V, Moraga F, Diaz-Elizondo J, Arredondo C, et al Ceramide-induced formation of ROS and ATP depletion trigger necrosis in lymphoid cells Free Radic Biol Med 2008 Mar 15; 44(6): 1146-1160 166 Amin H, Ergin M, Denning M, Quevedo M, Alkan S Characterization of apoptosis induced by protein kinase C inhibitors and its modulation by the caspase pathway in acute promyelocytic leukaemia Br J Haematol 2000 2000 Sep; 110(3): 552-562 167 Degterev A, Hitomi J, Germscheid M, Ch'en IL, Korkina O, Teng X, et al Identification of RIP1 kinase as a specific cellular target of necrostatins Nat Chem Biol 2008 May; 4(5): 313-321 168 Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E, et al A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles Cancer Res 2001 Jan 15; 61(2): 439444 144 169 Seglen PO, Gordon PB 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes Proc Natl Acad Sci U S A 1982 Mar; 79(6): 1889-1892 170 Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells Cell Struct Funct 1998 Feb; 23(1): 33-42 171 Murray D, Hill M, Nelson D Inhibitory action of sphingosine, sphinganine and dexamethasone on glucose uptake: studies with hydrogen peroxide and phorbol ester Life Sci 1990; 46(25): 1843-1849 172 Nelson D, Murray D Sphingolipids inhibit insulin and phorbol ester stimulated uptake of 2-deoxyglucose Biochem Biophys Res Commun 1986 1986 Jul; 138(1): 463-467 173 Lee YJ, Galoforo SS, Berns CM, Chen JC, Davis BH, Sim JE, et al Glucose deprivation-induced cytotoxicity and alterations in mitogenactivated protein kinase activation are mediated by oxidative stress in multidrug-resistant human breast carcinoma cells J Biol Chem 1998 Feb 27; 273(9): 5294-5299 174 Isaev NK, Stelmashook EV, Dirnagl U, Plotnikov EY, Kuvshinova EA, Zorov DB Mitochondrial free radical production induced by glucose deprivation in cerebellar granule neurons Biochemistry (Mosc) 2008 Feb; 73(2): 149-155 175 Jelluma N, Yang X, Stokoe D, Evan GI, Dansen TB, Haas-Kogan DA Glucose withdrawal induces oxidative stress followed by apoptosis in glioblastoma cells but not in normal human astrocytes Mol Cancer Res 2006 May; 4(5): 319-330 176 Meijer AJ, Codogno P AMP-activated protein kinase and autophagy Autophagy 2007 May-Jun; 3(3): 238-240 177 Cheng TJ, Wang YJ, Kao WW, Chen RJ, Ho YS Protection against arsenic trioxide-induced autophagic cell death in U118 human glioma cells by use of lipoic acid Food Chem Toxicol 2007 Jun; 45(6): 10271038 178 Yee KS, Wilkinson S, James J, Ryan KM, Vousden KH PUMA- and Bax-induced autophagy contributes to apoptosis Cell Death Differ 2009 Aug; 16(8): 1135-1145 179 Lavieu G, Scarlatti F, Sala G, Carpentier S, Levade T, Ghidoni R, et al Regulation of autophagy by sphingosine kinase and its role in cell survival during nutrient starvation J Biol Chem 2006 2006 Mar; 281(13): 8518-8527 145 180 Kim CH, Han SI, Lee SY, Youk HS, Moon JY, Duong HQ, et al Protein kinase C-ERK1/2 signal pathway switches glucose depletioninduced necrosis to apoptosis by regulating superoxide dismutases and suppressing reactive oxygen species production in A549 lung cancer cells J Cell Physiol 2007 May; 211(2): 371-385 181 Rego AC, Santos MS, Oliveira CR Influence of the antioxidants vitamin E and idebenone on retinal cell injury mediated by chemical ischemia, hypoglycemia, or oxidative stress Free Radic Biol Med 1999 Jun; 26(11-12): 1405-1417 182 Liu Y, Song XD, Liu W, Zhang TY, Zuo J Glucose deprivation induces mitochondrial dysfunction and oxidative stress in PC12 cell line J Cell Mol Med 2003 Jan-Mar; 7(1): 49-56 183 Kang D, Hamasaki N Mitochondrial oxidative stress and mitochondrial DNA Clin Chem Lab Med 2003 Oct; 41(10): 12811288 184 Behrend L, Henderson G, Zwacka RM Reactive oxygen species in oncogenic transformation Biochem Soc Trans 2003 Dec; 31(Pt 6): 1441-1444 185 Pelicano H, Carney D, Huang P ROS stress in cancer cells and therapeutic implications Drug Resist Updat 2004 Apr; 7(2): 97-110 186 Cheng CF, Juan SH, Chen JJ, Chao YC, Chen HH, Lian WS, et al Pravastatin attenuates carboplatin-induced cardiotoxicity via inhibition of oxidative stress associated apoptosis Apoptosis 2008 Jul; 13(7): 883-894 187 Husain K, Whitworth C, Hazelrigg S, Rybak L Carboplatin-induced oxidative injury in rat inferior colliculus Int J Toxicol 2003 Sep-Oct; 22(5): 335-342 188 Gewirtz DA A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin Biochem Pharmacol 1999 Apr 1; 57(7): 727-741 189 Maehara S, Tanaka S, Shimada M, Shirabe K, Saito Y, Takahashi K, et al Selenoprotein P, as a predictor for evaluating gemcitabine resistance in human pancreatic cancer cells Int J Cancer 2004 Nov 1; 112(2): 184-189 190 Donadelli M, Costanzo C, Beghelli S, Scupoli MT, Dandrea M, Bonora A, et al Synergistic inhibition of pancreatic adenocarcinoma cell growth by trichostatin A and gemcitabine Biochim Biophys Acta 2007 Jul; 1773(7): 1095-1106 146 191 Woiniak A, Drewa G, Wozniak B, Schachtschabel DO, MilaKierzenkowska C, Drewa T, et al The effect of antitumor drugs on oxidative stress in B16 and S91 melanoma cells in vitro Med Sci Monit 2005 Jan; 11(1): BR22-29 192 Irani K, Xia Y, Zweier JL, Sollott SJ, Der CJ, Fearon ER, et al Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts Science 1997 Mar 14; 275(5306): 1649-1652 193 Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, et al Cell transformation by the superoxide-generating oxidase Mox1 Nature 1999 Sep 2; 401(6748): 79-82 194 Carew JS, Zhou Y, Albitar M, Carew JD, Keating MJ, Huang P Mitochondrial DNA mutations in primary leukemia cells after chemotherapy: clinical significance and therapeutic implications Leukemia 2003 Aug; 17(8): 1437-1447 195 Hileman EO, Liu J, Albitar M, Keating MJ, Huang P Intrinsic oxidative stress in cancer cells: a biochemical basis for therapeutic selectivity Cancer Chemother Pharmacol 2004 Mar; 53(3): 209-219 196 Phillips DC, Allen K, Griffiths HR Synthetic ceramides induce growth arrest or apoptosis by altering cellular redox status Arch Biochem Biophys 2002 Nov 1; 407(1): 15-24 197 Ramsay E, Dos Santos N, Dragowska W, Laskin J, Bally M The formulation of lipid-based nanotechnologies for the delivery of fixed dose anticancer drug combinations Curr Drug Deliv 2005 2005 Oct; 2(4): 341-351 198 Maeda H The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting Adv Enzyme Regul 2001; 41: 189-207 199 Batist G, Gelmon KA, Chi KN, Miller WH, Jr., Chia SK, Mayer LD, et al Safety, pharmacokinetics, and efficacy of CPX-1 liposome injection in patients with advanced solid tumors Clin Cancer Res 2009 Jan 15; 15(2): 692-700 200 Tardi P, Johnstone S, Harasym N, Xie S, Harasym T, Zisman N, et al In vivo maintenance of synergistic cytarabine:daunorubicin ratios greatly enhances therapeutic efficacy Leuk Res 2009 Jan; 33(1): 129139 147 ... 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... (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