Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Farnaz Bahrami-Budlalu
A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy (PhD)
Cancer Research Laboratories, Department of Surgery, St George Clinical School, University of New South Wales
2015
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Acknowledgments
I am extremely grateful to my principal supervisor, Professor David L. Morris, for directing this project and supporting me throughout the PhD. I am very privileged to have been able to work under the guidance of someone so inspirational. I would also very much like to thank my research mentor and co- supervisor, Dr Mohammad H. Pourgholami, for his invaluable guidance and support during the last four years. His dedication to students covers not only scientific advice and methods, but also all the other elements required for being a qualified scientist. More than a mentor for research, he is also a great teacher who aided my personal growth during my PhD. I am indebted to him for the scientific training I have received. This thesis would not have been possible without the help from many individuals. Firstly, I would like to thank Dr Parvin Ataie-Kachoie and Dr. Ahmed H. Mekkawy for being the best colleagues and friends I could have ever wished. I would also like to thank Dr. Javed Akhtar, Mrs. Samina Badar, Dr. Zohra Ahmadi and Ms. Stephanie Chu for the enormous technical support they have offered me. I apologize to many of my colleagues and friends whose names cannot be all listed here. I want you to know that I would never have made it this far without your help and encouragement. Finally, I express my deepest gratitude to my father’s soul, my mother, my lovely sister, and my dear brother, for their support, encouragement, and unconditional love on which I have always relied. Moreover, I would like to thank my husband, Dr. Vahid Toloui and my daughter, Sara who have provided unwavering support and motivation to complete this thesis.
Page 1
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Table of Contents
Acknowledgments ...... 1
List of Figures ...... 6
List of Tables ...... 9
List of Abbreviations ...... 10
List of Publications ...... 14 Journal articles in preparation: ...... 15 Conference poster presentations ...... 15 Chapter 1. Literature Review ...... 17 1.1 Ovarian cancer ...... 17 1.1.1 Ovarian cancer cause and origin ...... 18 1.2 mTOR ...... 19 1.2.1 The mTOR proteins ...... 19 1.2.2 Regulation of mTORC1 Activity ...... 22 1.2.3 Downstream pathway of mTORC1 ...... 26 1.2.4 Cellular function of mTORC1 ...... 28 1.2.5 Regulator of mTORC2 Activity ...... 30 1.2.6 Downstream pathway of mTORC2 ...... 30 1.2.7 Cellular function of mTORC2 ...... 32 1.2.8 mTOR, diseases and the challenges associated with targeting mTOR ………………………………………………………………………….35 1.2.8.1 mTOR signalling and cancer ...... 35 1.2.8.2 Other mTOR-related disorders ...... 39 1.2.9 mTOR and autophagy ...... 39 1.2.10 mTOR and apoptosis ...... 40 1.2.11 mTOR inhibitors...... 41 1.2.11.1 First generation mTOR inhibitors: Rapamycin and its analogues……………………………………………………………………..41 1.2.11.2 Second generation mTOR inhibitors: Catalytic site (ATP- competitive) inhibitors ...... 44 1.3 Autophagy ...... 48 1.3.1 Molecular machinery of autophagy ...... 49 1.3.2 Regulation of autophagy ...... 53 1.3.3 Role of autophagy in physiology ...... 54 1.3.3.1 Autophagy-mediated protein quality control ...... 54 1.3.3.2 Autophagy-mediated organelle quality control ...... 55 1.3.3.3 Autophagy in cellular remodelling and development ...... 55 Page 2
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
1.3.3.4 Autophagy in metabolic homeostasis ...... 56 1.3.3.5 Autophagy confronts stress and environmental insults ...... 57 1.3.3.6 The importance of controlling p62/SQSTM1 levels by autophagy58 1.3.4 Autophagy and tumorigenesis ...... 60 1.3.4.1 Autophagy suppresses tumor initiation by limiting genome mutation ……………………………………………………………………….60 1.3.4.2 Autophagy suppresses tumor initiation and progression by limiting chronic inflammation ...... 61 1.3.4.3 Tumor cells with oncogenic mutations may be more dependent on autophagy for survival ...... 61 1.3.4.4 Autophagy inhibition sensitizes tumor cells to cell death ...... 62 1.4 Monepantel ...... 63 1.4.1 History ...... 63 1.4.2 Chemical structure ...... 64 1.4.3 Physicochemical properties ...... 65 1.4.4 Mode of action ...... 65 1.4.5 Pharmacokinetics of monepantel...... 66 1.4.6 Toxicology ...... 67 1.5 Aim of the study and hypothesis ...... 68 Chapter 2: Material and Methods ...... 70 2.1 Material ...... 70 2.2 Cell lines ...... 71 2.3 General methods ...... 72 2.3.1 Dissociation of cells from the culture flask ...... 72 2.3.2 Cell counting ...... 72 2.3.3 Freezing adherent cells ...... 73 2.3.4 Thawing of cryopreserved cells ...... 73 2.3.5 BCA protein assay ...... 73 2.4 In-vitro Methods ...... 74 2.4.1 Drug preparation ...... 74 2.4.2 Morphology ...... 75 2.4.3 Cell viability ...... 75 2.4.4 Cell proliferation assay (Sulforhodamine B, SRB) ...... 76 2.4.5 Cell proliferation assay (MTT) ...... 76 2.4.6 Colonogenic assay (colony formation) ...... 77 2.4.7 Cell cycle analysis ...... 77 2.4.8 Western blot analysis ...... 78 2.4.9 3H-thymidine incorporation assay ...... 79 2.4.10 Caspases activity assay ...... 80 2.4.11 Annexin V / 7-AAD staining ...... 81 2.4.12 Analysis of inter-nucleosomal DNA fragmentation (DNA Ladder) 81 2.4.13 Quantification of acidic vesicular organelles (AVO) by acridine orange (AO) staining ...... 82 2.4.14 Immuno-fluorescence, confocal scanning microscopy ...... 82 2.4.15 Enzyme-Linked Immunosorbent Assay (ELISA) for p70S6K and phosphorylated form ...... 83
Page 3
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
2.5 In-vivo method ...... 83 2.5.1 Drug formulation ...... 83 2.5.2 Mice ...... 84 2.5.3 Cell line ...... 84 2.5.4 Tumor induction ...... 84 2.5.5 Tumor treatment ...... 85 2.5.6 Evaluation of MPL formulation on tumor growth ...... 85 2.5.7 Sample collection and analysis...... 85 2.5.8 Immunohistochemistry ...... 86 2.5.9 Statistical analysis ...... 88 Chapter 3: Anticancer properties of aminoacetonitrile derivative, monepantel (ADD1566) in ovarian cancer ...... 89 3.1 Aim ...... 89 3.2 Results ...... 90 3.2.1 MPL decreases cell proliferation of ovarian cancer cells ...... 90 3.2.2 MPL decreases cell viability of ovarian cancer cells ...... 91 3.2.3 MPL decreases cell proliferation (based on their protein rate) of ovarian cancer cells ...... 93 3.2.4 MPL decreases cell metabolism of ovarian cancer cells ...... 95 3.2.5 MPL inhibits colony formation ...... 97 3.2.6 Antiproliferative activity of MPL is independent of acetylcholine signaling ...... 99 3.2.7 MPL induces G1 cell cycle arrest ...... 101 3.2.8 MPL down regulates the expression of cyclins and cyclin - dependent kinases to induce G1 cell cycle arrest ...... 105 3.2.9 MPL inhibits thymidine incorporation ...... 107 3.2.10 MPL induces PARP-1 cleavage...... 109 3.3 Discussion ...... 111 Chapter 4: Monepantel induces autophagy in human ovarian cancer cells through disruption of the mTOR/p70S6K signalling pathway ...... 114 4.1 Aim ...... 114 4.2 Results ...... 115 4.2.1 MPL reduced the expression of caspase 3 and 8 ...... 115 4.2.2 MPL decreased caspase-3 and -8 activation ...... 117 4.2.3 Cell death induced by MPL is not caspase-dependent ...... 119 4.2.4 MPL-induced cell death is not mediated via apoptosis ...... 121 4.2.5 MPL doesn’t induce DNA fragmentation ...... 125 4.2.6 MPL induces vacuole formation...... 127 4.2.7 Acidic vacuoles induced by MPL is concentration-dependent ... 129 4.2.8 MPL induces autophagy ...... 131 4.2.9 MPL regulates autophagy related proteins ...... 133 4.2.10 Inhibition of MPL-induced autophagy with 3-MA enhanced cell death ………………………………………………………………………..135 4.2.11 Inhibition of MPL-induced autophagy with wortmannin enhanced cell death in OVCAR-3 ...... 137
Page 4
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
4.2.12 Morphological study of inhibition MPL-induced autophagy with 3- MA …………………………………………………………………………..139 4.2.13 Inhibition of MPL-induced autophagy with CQ has no extra effect on cell death ...... 141 4.2.14 Morphological study of inhibition MPL-induced autophagy with CQ143 4.2.15 mTOR inhibitors sensitize ovarian cancer cells to MPL-induced cell death ...... 145 4.2.16 Morphological study of the effect of mTOR inhibitors on cells treated with MPL ...... 147 4.2.17 MPL suppresses mTOR ...... 149 4.2.18 MPL inhibits mTOR downstream mediators, p70S6K and 4E-BP1151 4.2.19 MPL inhibits p70S6K ...... 153 4.3 Discussion ...... 155 Chapter 5: Preclinical study of antitumor effects of the monepantel (ADD-1566) against human ovarian cancer cells in a xenograft model ... 158 5.1 Aim ...... 158 5.2 Results ...... 159 5.2.1 MPL impacts on subcutaneous tumor ...... 159 5.2.2 MPL induces necrosis of ovarian malignancy in-vivo ...... 161 5.2.3 MPL inhibits mTOR / p70S6K signalling pathway...... 163 5.2.4 MPL affects cell cycle regulators ...... 165 5.2.5 MPL induces cell cycle arrest through inhibition retinoblastoma protein (Rb) ...... 167 5.3 Discussion ...... 169 Chapter 6: General Discussion and Future Direction ...... 173
References ………………………………………………………………………..182
Page 5
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
List of Figures
Figure 1.1: A schematic presentation of mTOR structure Figure 1.2: Schematic representation of the mTOR complex components Figure 1.3: The mTOR signaling network Figure1.4: Structures of rapalogs Figure1.5: Machinery and regulators of autophagy Figure1.6: Regulation and functions of the autophagic pathway Figure1.7: Chemical structure of monepantel (MPL)
Figure 3.1: MPL inhibits growth of epithelial ovarian cancer cells Figure 3.2: Effect of MPL on cell viability is cell specific Figure 3.3: MPL suppresses proliferation (based on cell’s protein measurement) of ovarian cancer cells Figure 3.4: MPL suppresses metabolism of ovarian cancer cells Figure 3.5: MPL effects on the colony formation activity of ovarian cancer cells Figure 3.6: MPL-antiproliferative effect is not mediated through acetylcholine nicotinic receptor Figure 3.7: MPL effects on cell cycle progression of human ovarian cancer cells Figure 3.8: MPL modulates expression of the G1 cell cycle regulatory proteins Figure 3.9: MPL inhibits thymidine incorporation Figure 3.10: MPL induces PARP-1 cleavage
Figure 4.1: MPL reduced the expression of caspase 3 and 8 Figure 4.2: MPL decreased caspase-3 and -8 activation Figure 4.3: Cell death induced by MPL is not caspase-dependent Figure 4.4: MPL-induced cell death is not mediated via apoptosis
Page 6
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 4.5: MPL doesn’t induce DNA fragmentation Figure 4.6: MPL induces vacuole formation Figure 4.7: Acidic vacuoles induced by MPL is concentration-dependent Figure 4.8: MPL induces autophagy Figure 4.9: MPL regulates autophagy related proteins Figure 4.10: Inhibition of MPL-induced autophagy with 3-MA enhanced cell death Figure 4.11: Inhibition of MPL-induced autophagy with wortmannin enhanced cell death in OVCAR-3 Figure 4.12: Morphological study of inhibition MPL-induced autophagy with 3-MA Figure 4.13: Inhibition of MPL-induced autophagy with CQ has no extra effect on cell death Figure 4.14: Morphological study of inhibition of MPL-induced autophagy with CQ Figure 4.15: mTOR inhibitors sensitize ovarian cancer cells to MPL- induced cell death Figure 4.16: Morphological study of the effect of mTOR inhibitors on cells treated with MPL Figure 4.17: MPL suppresses mTOR Figure 4.18: MPL inhibits mTOR downstream, p70S6K and 4E-BP1 Figure 4.19: MPL suppresses p70S6K
Figure 5. 1: MPL reduces growth of ovarian cancer xenografts Figure 5. 2: MPL induces necrosis of ovarian malignancy, in-vivo
Figure 5. 3: MPL inhibits mTOR / p70S6K signalling pathway Figure 5. 4: MPL affects cell cycle regulators Figure 5. 5: MPL induces cell cycle arrest through inhibition retinoblastoma protein (Rb) Figure 5. 6: Flow diagram summarizing in-vivo experiments
Page 7
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Error! Reference source not found. Error! Reference source not found.
Page 8
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
List of Tables
Table 1.1: Pro-oncogenes and tumour suppressor genes linked to the mTOR pathway (Populo, Lopes, & Soares, 2012). Table 1.2: mTOR inhibitors in clinical trial
Table 2.1: List of antibodies used
Page 9
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
List of Abbreviations
3-MA 3-methyladenine
4E-BP1 4E-Binding Protein 1 A aa Amino acid AAD amino-acetonitrile derivative acr acetylcholine receptor Ade adenine AIF Apoptosis-Inducing Factor Akt protein kinase B (PKB) AMP Adenosine Monophosphate
AMPK adenosine monophosphate-activated protein kinase
Atg “autophagy-related” gene
ATM ataxia–telangiectasia mutated
ATP Adenosine Tri-Phosphate
ATR Rad3-related
B
bp, kb base pairs, kilo base pairs
BSA bovine serum albumin C oC degrees Celsius CDK Cyclin-Dependent Kinase CQ Chloroquine D
ddH2O deionised and distilled water
Page 10
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
DEG-3 C. elegans nAChR DES-2 C. elegans nAChR DEPC diethyl pyrocarbonate DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DNA-PK DNA-dependent protein kinase DTT dithiothreitol E EDTA ethylene diamine tetra-acetic acid (disodium) elF4E Eukaryotic Translation Initiation Factor 4E ER Endoplasmic reticulum ERK Extracellular Signal-Regulated Kinase F FAT named after FRAP, ATM and TRRAP FCS foetal calf serum FKBP12 FK506-binding protein 12 FOXO Forkhead box protein O FRAP FKBP-12-rapamycin associated protein FRB FKBP12–rapamycin-binding G g acceleration due to gravity (9.8 m.s-2) GAP GTPase Activating Protein GTP guanosine-triphosphate H h hours HEAT Huntingtin, Elongation Factor 3, A subunit of PP2A, TOR1 HDPE High-density polyethylene I IGF Insulin-like growth factor IRS-1 Insulin receptor substrate 1
Page 11
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
K kDa Kilo dalton L LC3 Light Chain 3 LKB1 Liver Kinase B1 M MAP kinase mitogen-activated protein" kinase min minutes MPL monepantel mLST8 mammalian lethal with SEC13 protein 8 mSIN1 mammalian stress-activated protein kinase interacting protein 1 mTOR mammalian target of rapamycin
µL, mL, L microliter, millilitre, litre µM, mM, M micromolar, millimolar, molar N NF-κB Nuclear Factor Kappa B NSCLC Non-Small Cell Lung Carcinoma O OD optical density P p62/SQSTM1 P62 mammalian sequestrome-1 p70S6K phosphoprotein 70 ribosomal protein S6 kinase PARP Poly ADP (Adenosine Diphosphate)-Ribose Polymerase PBS phosphate buffered saline PDK1 Phosphoinositide-dependent kinase 1 PI3K Phosphoinositide 3-Kinase PIKK phosphoinositide 3-kinase (PI3K)-related protein kinase PIP3 phosphatidylinositol-triphosphate PP2A protein phosphatase 2A
Page 12
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
PRR5 Proline-rich protein-5 PTEN phosphatase and tensin R RAD001 Everolimus Rheb Ras homolog enriched in brain RNA ribonucleic acid rpm revolutions per minute RSK1 Ribosomal S6 Kinase 1 S s seconds SC Subcutaneous SDS sodium dodecyl sulfate Ser Serine SMG-1 Suppressor of morphogenesis in genitalia-1 T Thr. Threonine TOR targets of rapamycin TOS Tor signalling sequence Tris [2-amino-2-hydroxy-(hydroxymethyl)-propane-1, diol, (tris)] TSC Tuberous sclerosis complex U u unit ULK1 Unc-51 Like Kinase 1 UV ultra violet UVRAG UV Radiation Resistance Associated Gene V v/v, w/v volume per volume, weight per volume VEGF vascular endothelial growth factor Vps34 vacuolar protein sorting 34
Page 13
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
List of Publications
Published Journal articles:
1- Farnaz Bahrami, Mohammad H. Pourgholami*, David L Morris, Tetracyclines: drugs with huge therapeutic potential, Mini-Reviews in Medicinal Chemistry, 2012; 12, 1-9
2- Farnaz Bahrami, Parvin Ataie-Kachoie, Mohammad H. Pourgholami, David L. Morris, p70 Ribosomal protein S6 kinase (Rps6kb1): an update, Journal of Clinical Pathology, 2014 Aug 6. pii: jclinpath-2014-202560. doi: 10.1136/jclinpath-2014-202560
3- Farnaz Bahrami, David L. Morris, Lucien Rufener, Mohammad H. Pourgholami, Anticancer properties of novel aminoacetonitrile derivative monepantel (ADD 1566) in pre-clinical models of human ovarian cancer, American Journal of Cancer Research, 2014; 4(5): 545–557
4- Farnaz Bahrami, David L. Morris, Ahmed Mekkawy, Lucien Rufener, Mohammad H. Pourgholami, Monepantel induces autophagy in human ovarian cancer cells through disruption of the mTOR / p70S6K signalling pathway, American Journal of Cancer Research, 201; 4 (5): 558-71
5- Parvin Ataie-Kachoie, Farnaz Bahrami, David L. Morris, Mohammad H. Pourgholami, Minocycline attenuates hypoxia-inducible factor-1α expression correlated with modulation of p53 and AKT/mTOR/p70S6K/4E-BP1 pathway in ovarian cancer: in vitro and in vivo studies, Accepted, American Journal of Cancer Research, Jan 2015
Page 14
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Journal articles in preparation:
1- Farnaz Bahrami, Parvin Ataie-Kachoie, David L. Morris, Ahmed Mekkawy, Mohammad H. Pourgholami, In-vivo antitumor effects of the monepantel (ADD-1566) against human ovarian cancer cells in a xenograft model
2- Farnaz Bahrami, David L. Morris, Mohammad H. Pourgholami, mTOR, an update review
3- Farnaz Bahrami, Hieng Chiong Lu, Parvin Ataie-Kachoie, David L. Morris, Mohammad H. Pourgholami, Monepantel: a novel anthelmintic with potential anticancer activity
Conference poster presentations
1- Farnaz Bahrami, Mohammad H. Pourgholami, David L. Morris: Effect of minocycline on ATP levels in ovarian cancer cells, presented at St. George and Sutherland Medical Research Symposium, Sydney, Australia, Oct 2011
2- Farnaz Bahrami, Mohammad H. Pourgholami, David L. Morris: Monepantel suppress ovarian cancer cell proliferation by arresting G0-G1 cell cycle and down regulating cyclin A and E, Cdk 2 and 4, presented at St. George and Sutherland Medical Research Symposium, Sydney, Australia, Oct 2012
3- Farnaz Bahrami, Mohammad H. Pourgholami, David L. Morris: Monepantel suppress ovarian cancer cell proliferation by arresting cell cycle at G1 through AMPK/ ERK / p70S6k pathway and down regulating cyclin D1, E, Cdk 4 and 2, presented at Lorne Cancer Conference, Melbourne, Australia, Feb. 2013
Page 15
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
4- Farnaz Bahrami, Mohammad H. Pourgholami, David L. Morris: Monepantel (AAD-1566) mediates caspase-independent cell death along with autophagy in ovarian cancer cells, St. George and Sutherland Medical Research Symposium, Sydney, Australia, Oct 2014
5- Farnaz Bahrami, Mohammad H. Pourgholami, David L. Morris: Monepantel (AAD-1566) inhibits mammalian target of rapamycin signaling through inhibition of major components in the mTOR axis and induction of autophagy in ovarian cancer cells, 15th Biennial Meeting of the International Gynecologic Cancer Society, Nov. 2014
6- Farnaz Bahrami, Mohammad H. Pourgholami, David L. Morris: A novel aminoacetonitrile derivative, monepantel (AAD-1566) inhibits ovarian cancer cell growth through suppression mTORC1 and induction of autophagy, Sydney Cancer Conference, Nov. 2014
Page 16
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
CChhaapptteerr 11.. Literature Review
1.1 Ovarian cancer
Ovarian cancers pose the greatest challenge for gynaecological oncology. It is the sixth most common cancer among women. Worldwide there are more than 200,000 new cases of ovarian cancer each year, accounting for around 4% of all cancers diagnosed in women with approximately 6.6 new cases per 100,000 women per year (Al Rawahi et al., 2013). This malignancy is characterized by the highest mortality rate among Gynecologic malignancies (Clarke-Pearson, 2009). It has a disproportionately high mortality rate, with only 30% of women surviving 5 years after diagnosis (Buys et al., 2011). The high death-rate is related to the difficulty of detecting it at an early stage (75% of cases diagnosed in advanced stages) (Torpy, Burke, & Golub, 2011), coupled with its non-specific signs and symptoms, the lack of specific sets of markers and sensitive diagnostic tools, the lack of effective therapies for advanced disease, as well as relapse as a result of expansion of chemo-resistant clones (Gubbels, Claussen, Kapur, Connor, & Patankar, 2010).
Effective treatment protocols for ovarian cancer are limited (Gubbels et al., 2010; Hennessy, Coleman, & Markman, 2009). Standard treatment of ovarian cancer involves surgical management with staging or debulking surgery and chemotherapy with a platinum and taxane-containing regimen (Han, Wakabayashi, & Leong, 2013). In spite of advances in surgical and cytotoxic chemotherapy management, survival for patients with advanced disease remains low. Even when complete remission is achieved following optimum first-line therapy, around 80% of patients will develop recurrent disease
Page 17
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
(Hennessy et al., 2009; Mitchell et al., 2010). Therefore, there is a growing need for novel therapies and techniques to improve the treatment of ovarian cancer.
1.1.1 Ovarian cancer cause and origin
Ovarian cancer comprises a heterogeneous group of neoplasms, exhibiting a wide range of morphological characteristics, genetic alterations, and biological behaviours. There are three major types of ovarian cancer: epithelial, germ cell and sex-cord stromal, although epithelial ovarian cancer accounts for 90% of all ovarian cancers. Morphologically, epithelial ovarian cancer can be subdivided into serous, mucinous, endometrioid, clear cell and transitional subtypes (Lee et al., 2007; Nezhat et al., 2008). The origin of epithelial ovarian cancer is still open for debate; obvious sites include the ovarian surface epithelium and post- ovulatory inclusion cysts, although non-ovarian structures such as the peritoneum, Fallopian tube and endometriotic lesions have all been proposed (Ricciardelli & Oehler, 2009).
The etiology of epithelial ovarian cancer is poorly understood. In an effort to identify the causes of this deadly malignancy, a few hypotheses have been put forward. These include the incessant ovulation hypothesis (Fathalla, 1971), the gonadotropin hypothesis (Cramer & Welch, 1983) and the hormonal hypothesis (Risch, 1998). In these theories, continuous damage and repair of the ovarian surface epithelia during ovulation and excessive exposure of the ovarian surface epithelia to gonadotropins or sex hormones have been attributed to the formation of ovarian cancer. Studies have shown that a panel of inflammatory modulators are activated during cyclical ovulation (Espey, 1994). Similarly, elevation of estrogens and androgens (Orvieto, Fisch, Yulzari-Roll, & La Marca, 2005) amplifies immune responses by recruiting pro-inflammatory cells and molecular effectors. Therefore, these hypotheses collectively suggest that normal physiological activities of the ovary are accompanied by general activation of inflammatory mediators, which may cause ovarian tumorigenesis. This further strengthens the hypotheses, explaining that in the pathogenesis of
Page 18
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
ovarian tumors, chronic inflammation seems to play a significant role. Additionally this hypothesis is supported by indirect epidemiological and clinical evidence linking ovarian cancer with pelvic inflammatory disease, endometriosis or polycystic ovary syndrome (Kisielewski, Tolwinska, Mazurek, & Laudanski, 2013).
1.2 mTOR
1.2.1 The mTOR proteins
The TOR proteins are a family of serine/threonine protein kinases that include ataxia–telangiectasia mutated (ATM), Rad3-related (ATR), DNA- dependent protein kinase (DNA-PK), and suppressor of morphogenesis in genitalia-1 (SMG-1) protein kinase. These kinases are characterized by large size (>2,500 amino acid) and a C-terminally located kinase domain (Abraham, 2004). The C-terminal kinase domains are similar to the kinase domain of phosphoinositide-3-kinase (PI3K), and from here, they get the name PI3K- related kinases (PIKKs). The N-terminus possesses 20 tandem HEAT domains, which are named based on their presence in Huntingtin protein, Elongation factor 3, the A subunit of PP2A and TOR1.
mTOR is an atypical serine/threonine protein kinase with a molecular weight of 290 kDa. Structurally, the N-terminus of mTOR consists of 20 tandemly repeated motifs (HEAT motifs).
The C-terminus consists of mutated FRAP-ataxia-teleangiectasia (FAT, FRAP) domain, a transformation/transcription domain-associated protein domain, an FKPB12-rapamycin-binding (FRB) domain, a catalytic kinase domain, and a FAT carboxy-terminal domain (FAT C-terminus, FATC). It is speculated that the HEAT repeats serve to mediate protein-protein interactions, the FRB domain as suggested by its name provides a docking site for the
Page 19
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
FKBP12/rapamycin complex, and the FAT and FATC domains modulate mTOR kinase activity via unknown mechanisms (Q. Yang & Guan, 2007) (Figure 1.1).
Figure 1.1: A schematic presentation of mTOR structure Schematic of mTOR complex components. HEAT: a protein-protein interaction structure of two tandem anti-parallel a-helices found in huntingtin, elongation factor 3, PR65/A and TOR; FAT: a domain structure shared by FRAP, ATM and TRRAP, all of which are PIKK family members; FRB: FKBP12/rapamycin binding domain; FATC: FAT C-terminus. Adapted from (Benjamin, Colombi, Moroni, & Hall, 2011).
Page 20
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
The mTOR pathway is a key regulator of cell growth and proliferation and increasing evidence suggests that its deregulation is associated with human diseases, including cancer and diabetes (Pavlidou & Vlahos, 2014; Sarbassov, Ali, & Sabatini, 2005). The mTOR signaling pathway is considered the central regulator of ribosome biogenesis, protein synthesis, and cell growth.
mTOR is found in two structurally and functionally distinct complexes to regulate growth and metabolism. In mammals, the mTOR complex 1 (mTORC1) contains mTOR, mLST8 (G protein beta protein subunit-like, GβL), PRAS40 (a proline-rich Akt substrate of 40 kDa, a raptor-interacting protein), raptor (regulatory associated protein of mTOR), and deptor (mTOR-interacting protein, inhibitory protein). mTORC1 is sensitive to the immunosuppressive and anticancer drug rapamycin.
mTORC2 contains mTOR, mLST8, rictor (rapamycin-insensitive companion of mTOR, also known as the mammalian homolog of AVO3P, mAVO3), mSIN1 (mammalian stress-activated protein kinase interacting protein 1, MIP1, mAVO1, the mammalian homolog of Avo1p, necessary for mTORC2 assembly and Akt/PKB phosphorylation), PRR5 (proline-rich protein 5, belongs to the small family of pseudo-response regulators (PRRs)) and deptor. The mTORC2 complex is insensitive to rapamycin (Bai & Jiang, 2010; T. R. Peterson et al., 2009; H. Yang et al., 2013) (Figure 1.2).
The two complexes signal via different effector pathways to control distinct cellular processes. The mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids and is deregulated in common cancers. The mTORC2 complex phosphorylates Akt at Ser473 and regulates the actin cytoskeleton (Bracho-Valdes et al., 2011; Jacinto et al., 2004).
Page 21
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 1.2: Schematic representation of the mTOR complex components The mTORC1 complex consists of mTOR, mLST8, PRAS40 and raptor and the mTORC2 complex consists of mTOR, mLST8, rictor, mSIN1, and PRR5.
1.2.2 Regulation of mTORC1 Activity
1.2.2.1 Activation of mTORC1 by the PI3K signaling pathway
The PI3K pathway has a critical role in aggressive tumorigenesis. PI3K signaling is activated by various extracellular signals including peptide growth factors such as insulin andIGFs. The PI3K activity that results in PIP3 production is tightly controlled and negatively regulated by several phosphatases. The PTEN (phosphatase and tensin homolog on chromosome 10) lipid phosphatase dephosphorylates PIP3 at the 3’ position, whereas SHIP- 1 phosphatase dephosphorylates it at the 5’ position, in both cases limiting the production of PIP3. Genetic inactivation of PTEN by mutation, leads to constitutive activation of the PI3K/AKT/TSC2/mTORC1 cascade (Fruman & Rommel, 2014; Hay & Sonenberg, 2004).
Page 22
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Akt is a serine/threonine kinase, also known as protein kinase B, a critical downstream effector of PI3K. Mammalian cells express three Akt proteins encoded by different genes. Full activation of Akt requires Akt phosphorylation at Ser473 and Thr308 by PI3K and PDK1 (the phosphoinositide-dependent protein kinase), respectively. Akt phosphorylates TSC2, destabilizes it and disrupts its interaction with TSC1 leading to activation of mTOR (Bai & Jiang, 2010) (Figure 1.3).
1.2.2.2 Inhibition of mTOR by the LKB1/AMPK/TSC2 signaling pathway
The serine/threonine kinase LKB1 is a tumor suppressor gene. LKB1 is a central regulator of cell polarity and energy metabolism through its capacity to activate adenine monophosphate-activated protein kinase (AMPK). AMPK is activated in response to ATP depletion or increased AMP levels. Activation of AMPK phosphorylates and activates TSC2 inducing mTOR down regulation (Bai & Jiang, 2010; Meric-Bernstam & Gonzalez-Angulo, 2009) (Figure 1.3).
1.2.2.3 Inhibition of mTOR by the tuberous sclerosis complex
(TSC1/TSC2)
Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs upon mutation of either the TSC1 or TSC2 gene, which encodes Hamartin or Tuberin, respectively. The TSC1/TSC2 (TSC1/2) has been known as the major upstream inhibitory regulator of mTOR. TSC2 protein contains a GAP homology domain at its Cterminus. In vitro, TSC2 stimulates GTP hydrolysis of Rheb. TSC1 does not have any GAP activity and is not required for TSC2/GAP activity towards Rheb in vitro. Rheb (Ras homolog enriched in brain) is a member of the Ras family proteins. Rheb, a small GTPase that belongs to a unique family within the Ras superfamily of GTPase. The small GTPase Rheb is a positive upstream regulator of the target of mTORC1. TSC2 regulates Rheb-GTP levels. TSC2 acts as a GTPase-activating protein (GAP) for Rheb. Therefore, TSC2 inhibits Page 23
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Rheb activity. Rheb in its active GTP-bound state (Rheb-GTP) binds to and activates mTORC1. TSC1/2 inhibits mTORC1 activity by limiting the amount of GTP-bound Rheb available to stimulate mTORC1 (Bai & Jiang, 2010; Hay & Sonenberg, 2004).
Multiple signaling cascades converge on TSC2, leading to its phosphorylation and inactivation. TSC2 is phosphorylated by multiple kinases, including Akt, RSK1, ERK, and AMPK (Bai & Jiang, 2010) (Figure 1.3). Active Akt phosphorylates TSC2 directly on multiple sites (Ser939, Ser981, and Thr1462). Phosphorylation of TSC2 inactivates the GTPase activator domain function of TSC2, disrupts the TSC1/2 complex and stimulates activity of Rheb and mTOR (Tee, Manning, Roux, Cantley, & Blenis, 2003).
Under energy starvation conditions, the AMP-activated protein kinase (AMPK) phosphorylates TSC2 at Thr1227 or Ser1345. The phosphorylation of TSC2 at these sites by AMPK improves the ability of TSC2 to inhibit mTOR activity by activating GAP activity of TSC2 (Inoki, Zhu, & Guan, 2003). ERK, and RSK directly phosphorylate TSC2 at Ser664 and Ser1798 resulting in inhibition of TSC2 function (Bai & Jiang, 2010).
Page 24
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 1.3: The mTOR signaling network mTOR complexes and mTOR signaling network. Selected components and functions of both mTOR1 and mTORC2 are indicated, and both mTORC1 and mTORC2 additionally interact with deptor, which usually inhibits the activity of both complexes. Growth factor such as insulin stimulates mTORC1 (and probably mTORC2), leading to Akt activation to inhibit TSC2, a GTPase activating protein for Rheb. Amino acid also activates mTORC1 through glutamine-leucine and Rag–Regulator complex, which is required for full activation of mTORC1 by growth factor. Little is known about activation mechanism of mTORC2. Feedback loop by S6K-IRS or S6K-Rictor exists in mTOR signaling. In contrast, cellular stress activates TSC2 and inhibits mTOR pathway. Adapted from: (Watanabe, Wei, & Huang, 2011).
Page 25
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
1.2.3 Downstream pathway of mTORC1
The downstream signals of mTORC1 are characterized by two independent targets, p70S6K and 4EBP1/eIF4E complex.
1.2.3.1 mTOR / p70S6K pathway
The p70S6K is a major downstream effector of the mammalian target of rapamycin. The p70S6K is a mitogen-activated serine/threonine kinase, which plays a crucial role in the control of the cell cycle (during progression through the G1 phase), of growth and survival. The p70S6K phosphorylates the 40S ribosomal protein S6, leading to up regulation of translation and protein synthesis. The p70S6K is regulated by diverse extracellular signals. The activity of p70S6K is controlled by multiple phosphorylation events located within the catalytic, linker and pseudosubstrate domains. Activation of p70S6K depends on the level of its phosphorylation state at eight sites: Thr229, Ser371, Thr389, Ser404, Ser411, Ser418, Thr421, and Ser424.
Phosphorylation of Thr229 in the catalytic domain and Thr389 in the linker domain are most critical for the kinase function. The mTORC1 complex phosphorylates p70S6K at Thr389 and Ser371. PDK1 binds to and phosphorylates p70S6K at Thr229 (Dufner & Thomas, 1999; Pearson et al., 1995; Pullen & Thomas, 1997). The carboxyl terminus of p70S6K has a set of Ser and Thr residues (Ser411, Ser418, Ser424, and Thr421), which might be phosphorylated by the MAP kinases ERK1/2 and p38 (B. F. Bahrami, Ataie- Kachoie, Pourgholami, & Morris, 2014; Mukhopadhyay et al., 1992; Su & Jacinto, 2011).
1.2.3.2 mTOR / 4E-BP1 pathway
The 4E-BP1 is a downstream component of the mTOR pathway. 4E-BP1 is a protein identified as a repressor of the cap-binding protein (eIF4E). 4EBP1 binds to eIF4E, which prevents the formation of the active eIF4F complex
Page 26
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
(eIF4A-eIF4G-eIF4E complex). Hyperphosphorylation of 4EBP1 by mTOR, results in the release of eIF4E, thus, allowing the translation complex to assemble (Hay & Sonenberg, 2004). Down regulation of mTOR induces hypophosphorylation of 4E-BP1 leading to 4E-BP1 binding to eIF4E, and inhibition of cap-dependent translation.
Eukaryotic translation initiation factor 4E (eIF4E), the mRNA 5'-cap-binding protein, is central component in the initiation and regulation of translation in eukaryotic cells. EIF4E binds to the mRNA cap structure to mediate the initiation of translation. The level of free eIF4E might be raised due to increased eIF4E expression or increased phosphorylation and expression of 4EBP1 (De Benedetti & Graff, 2004).
Cyclin D1 is considered the prime downstream target protein for eIF4E dependent protein translation. Expression of eIF4E significantly correlates with increased cyclin D1 protein translation. eIF4E enhances nuclear export of cyclin D1 mRNAs (Hershey, 2010; Robert & Pelletier, 2009).
Additionally, it has been shown that inhibition of mTOR signaling was associated with reduction of cyclin D1 expression (Averous, Fonseca, & Proud, 2008; Dong et al., 2005; Yu, Shen, Khor, Kim, & Kong, 2008). Cyclin D1 is a key regulator of the G1 phase of the cell cycle, which drives cells through the G1/S phase transition (Stacey, 2003). Down regulation of cyclin D1 function results in cell cycle arrest in G0/G1 cell cycle phase.
1.2.3.3 Regulation of protein serine/threonine phosphatase
mTOR directly phosphorylates p70S6K and 4EBP1, and also indirectly increases their phosphorylation by inhibition of protein serine/threonine phosphatase (Dufner & Thomas, 1999).
Page 27
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
The two main classes of serine/threonine protein kinases, PP1 and PP2A, are extensively involved in many signaling pathways. Treatment of cells with phosphatase inhibitors calyculin A prevents 4EBP1 dephosphorylation. At the same time, inhibition of p70S6K activity by rapamycin, curcumin or amino acid deprivation requires phosphatase activity (Yu et al., 2008). The serine/threonine phosphatase PP2A is a prime candidate for such a mTORdependent phosphatase. PP2A dephosphorylates p70S6K in vitro and associates with full- length p70S6K, rather than the rapamycin-resistant N- and C-terminal truncated p70S6K mutant (R. T. Peterson, Desai, Hardwick, & Schreiber, 1999).
1.2.4 Cellular function of mTORC1
1.2.4.1 Protein synthesis
Downstream of PI3K-Akt activation, mTORC1 plays major role in the phosphorylation of proteins that regulate or are involved in mRNA translation. As mentioned before, p70S6K and 4E-BP1 are the major direct targets of mTORC1 in this process. After activation by mTORC1, S6K phosphorylates several proteins that are associated with mRNA translation such as ribosomal protein S6, eIF4B, S6K1 aly/REF-like target (SKAR), programmed cell death 4 (PDCD4) and eukaryotic elongation factor 2 kinase (eEF-2k) (Ma & Blenis, 2009). Phosphorylation of 4E-BP on the other hand, prevents its binding with eIF-4E and thereby increases protein translation. mTORC1 increases translation of a subset of mRNA that contains 5’ tract of oligopyrimidine (TOP). 5’ TOP mRNA encodes components of the translation apparatus (reviewed in (Sengupta, Peterson, & Sabatini, 2010). A novel example of translational control by mTOR has been shown that involves IRES- (internal ribosome entry segment) driven translation of specific mRNAs (Ramirez-Valle, Badura, Braunstein, Narasimhan, & Schneider, 2010).
Page 28
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
1.2.4.2 Ribosome biogenesis
Ribosome biogenesis is a high energy-demanding process, and mTOR tightly controls this process based upon nutrient and energy availability. The synthesis of ribosomal proteins and rRNA is positively regulated by mTORC1. Studies with rapamycin shown that mTOR inhibits transcription of RNA polymerase I (Pol I)-dependent rRNA genes, Pol II-dependent ribosomal protein genes, and Pol III-dependent tRNA genes, which altogether block ribosome biosynthesis. mTORC1 upregulates transcriptional activity of the rRNA polymerase RNA polymerase I (RNAPI) through S6K1 (Mayer, Zhao, Yuan, & Grummt, 2004) and regulates processing of 35s and 5s rRNA (D. E. Martin & Hall, 2005).
1.2.4.3 Metabolism mTORC1 regulates several metabolic pathways by controlling key steps at the transcriptional, translational and post-translational level in different tissues types (reviewed in (Polak & Hall, 2009). Active mTORC1 promotes expression of hypoxia-inducible factor-1 (HIF-1), mostly by regulating translation of its alpha subunit through 4E-BP1, which activates the transcription of many genes involved in cellular metabolism (Majmundar, Wong, & Simon, 2010). Studies showed that mTORC1 regulates glycolysis, sterol and lipid biosynthesis in addition to control key steps in the pentose phosphate pathway (Duvel et al., 2010).
1.2.4.4 Autophagy
Autophagy is a catabolic process that recycles cellular organelles and proteins. It functions as a quality control, and a mechanism by which cells replenishes their intracellular nutrients content under conditions of poor nutrient availability. Under nutrient-rich conditions, mTORC1 maintains low levels of autophagy. It inhibits a conserved protein complex containing the protein kinases Atg1 and Atg2 that are required for induction of autophagy (Neufeld, 2010). It has been shown that TORC1 directly phosphorylates Atg13 (ULK1) at multiple serine residues. Atg13 is an essential regulatory component of autophagy upstream of Page 29
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
the Atg1 kinase complex and in its phosphorylated state directly inhibits the Atg1-driven autophagic process (Kamada et al., 2010).
1.2.5 Regulator of mTORC2 Activity
Compared to mTORC1, which is activated by several factors as described above much less is known about the upstream regulators of mTORC2. Growth factors have been shown to activate mTORC2 signaling as demonstrated by Akt phosphorylation at Ser473 (Frias et al., 2006; Sarbassov et al., 2006). Gan et al showed that addition of PIP3 to a mTORC2 kinase assay enhances Akt phosphorylation in vitro (Gan, Wang, Su, & Wu, 2011). mTORC2, by associating with translating ribosomes, phosphorylates Akt at Thr450 site and phosphorylation at this site is not inducible by growth factors (Oh et al., 2010). The TSC1-TSC2 complex that downregulates mTORC1 is required for proper activation of mTORC2. TSC1-TSC2 complex directly associates with mTORC2 via rictor, and regulation of mTORC2 activity by this complex appears to be independent from its GTPase activity towards Rheb (Huang, Dibble, Matsuzaki, & Manning, 2008).
1.2.6 Downstream pathway of mTORC2
Studies have shown that mTORC2 recognizes and phosphorylates AGC kinases, which play key roles in multiple intracellular signaling pathways. The AGC kinases are Ser/Thr kinases from a large family of conserved proteins. The prototypical members of the AGC kinase family are cAMP-dependent protein kinase (PKA), protein kinase G (PKG), and protein kinase C (PKC). In addition, the family also includes Akt, S6K and serum and glucocorticoidinduced kinase (SGK). mTORC2 phosphorylates AGC kinases at their turn motif (TM) having specific Thr-Pro-Pro sequence and at hydrophobic motif (HM) when have specific Ser/Thr-Tyr/Phe sequence. Phosphorylation by mTORC2 allosterically activates AGC kinases in addition to catalytic activation of the
Page 30
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
kinases by PDK1 (reviewed in (Oh & Jacinto, 2011)). The substrates known for mTORC2 are described hereafter.
1.2.6.1 Akt / PKB
Akt is phosphorylated at two sites by mTORC2; firstly at Thr450 of the TM and secondly at Ser473 of the HM domain. TM phosphorylation of Akt is a one-time irreversible step that occurs exclusively during the synthesis of nascent Akt, when the polypeptide is still attached to the ribosome. TM phosphorylation is essential for Akt stability and its deficiency results in co-translational ubiquitination of nascent Akt (Oh et al., 2010; Rosse et al., 2010). Thr450 phosphorylation of Akt at TM is solely done by mTORC2 and this is well conserved from yeast to human (Rosse et al., 2010). In contrast, phosphorylation at HM Ser473 is a post-translational modification that occurs at the membrane where mTORC2 is supposed to co-localize with Akt. Growth factors and hormones induce HM Ser473 phosphorylation that allosterically activates Akt, thereby increasing its activity towards many of its substrates such as forkhead box O1/3 (FoxO1/3). Studies have shown that HM phosphorylation by mTORC2 gives substrate specificity to Akt, as mTORC2-deficient cells showed defective FoxO1/3 phosphorylation but had normal GSK3 and TSC2 phosphorylation (Guertin et al., 2006; Jacinto et al., 2006).
1.2.6.2 PKC
PKCs are conserved signaling molecules with a variety of cellular functions, mainly responsible for distribution of signals (Rosse et al., 2010). Phosphorylation at the HM Ser657/660 of all conventional PKCs (cPKC) and of some novel PKC (nPKC), as well phosphorylation of PKC/II TM at Thr638/641, requires mTORC2. mTORC2 was shown to be engaged in PKC maturation and stability like Akt (reviewed in (Oh & Jacinto, 2011)). Introduction 28 SIN1 deletion significantly decreases cPKC expression levels as TM phosphorylation is abolished. This is in contrast with Akt, which maintains its
Page 31
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
level with the help of Hsp90- induced TM phosphorylation of Akt (Ikenoue, Inoki, Yang, Zhou, & Guan, 2008).
1.2.6.3 SGK mTORC2 is required for Ser422 phosphorylation present in HM of SGK. SGK is a short-lived protein stimulated by growth factors and conditions of osmotic stress (Lang et al., 2006). Unlike Akt and cPKC, SGK1 levels are not decreased in mTORC2-deficient cells, but rather increased in rictor null cells. Disruption of mTORC2 leads to decreased SGK1 phosphorylation, which in turn affects activation of its specific substrate NDRG1 (N-myc downregulated gene 1) (Garcia-Martinez & Alessi, 2008). Protor-1 functions as the adaptor of mTORC2 activity to phosphorylate HM of SGK1 (Pearce, Sommer, Sakamoto, Wullschleger, & Alessi, 2011). A couple of years ago, mTORC2 has been shown to activate epithelial sodium channel (ENaC)-dependent sodium transport in kidney cells through phosphorylation of SGK1 (Lu et al., 2010).
1.2.7 Cellular function of mTORC2
1.2.7.1 Cytoskeleton organization
Early studies by Loewith and colleagues have shown that TORC2 controls cell cycle-dependent polarization of the actin cytoskeleton in yeast via the activation of Rho1 GTPase switch (Jacinto et al., 2004). mTORC2 activates PKC by phosphorylating its HM site, which causes its interaction with small GTPase Rho and Rac and thereby it signals to actin cytoskeleton (Jacinto et al., 2004; Sarbassov et al., 2004). Knockdown of mTOR, rictor, mLST8, but not raptor causes defective actin reorganization along with decreased Rac1 activation, upon serum restimulation in mammalian cells. Rac1 was found to be a part of both mTOR complexes upon growth factor stimulation. Activated Rac1 could mediate actin rearrangement by translocating to plasma membrane, where it
Page 32
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
increases PIP3 synthesis and thereby could led to actin organization (Saci, Cantley, & Carpenter, 2011).
1.2.7.2 Protein synthesis and maturation
Protein synthesis is a process mainly associated with mTORC1, but different studies have suggested a role for mTORC2 in this basic cellular process. Intact mTORC2 localizes in polysome fractions and directly interacts with the 60S large ribosome subunit. In particular, rictor, which is located at the exit tunnel of ribosomes, can form stable interactions with the ribosomal proteins L23a and L26 (Oh et al., 2010; Zinzalla, Stracka, Oppliger, & Hall, 2011). The nature of this interaction plays a role in mTORC2-mediated co-translational maturation of the nascent Akt polypeptide (Oh et al., 2010). Zinzalla and coworkers indentified a protein involved in ribosome biogenesis and rRNA maturation using yeast genetic screens, NIP7, which regulates mTORC2 activity. This suggests that association of mTORC2 with assembled ribosomes or ribosomal proteins activates mTORC2. Upon mTORC2 inhibition or disruption, total translation and polysomes were shown to be more attenuated compared to rapamycin treatment, which inhibits mTORC1, suggesting a role of mTORC2 in translation (Oh et al., 2010; Zinzalla et al., 2011).
1.2.7.3 Chemotaxis, proliferation and survival
In line with the early demonstration of its role in actin reorganization, mTORC2 was shown to be involved in cell migration and cancer metastasis (Gulhati et al., 2011). Rictor interacts with PKC and regulates metastasis of breast cancer cells, where rictor acts as a mediator of chemotaxis (F. Zhang et al., 2010). In normal cell types such as neutrophils, chemotaxis is regulated by mTORC2 via activation of adenylyl cyclase 9 (AC9) (L. Liu, Das, Losert, & Parent, 2010). It has been reported that rictor/mTORC2 are to be essential for maintaining a balance between -cell proliferation and cell size (Gu, Lindner, Kumar, Yuan, & Magnuson, 2011), whereas in TSC-null cells, mTORC2 modulates its
Page 33
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
proliferation and survival through RhoA GTPase and Bcl2 proteins (Goncharova et al., 2011).
1.2.7.4 Glucogenesis
Gluconeogenesis is the process of biosynthesis of new glucose. In a study by Wang and colleagues suggests a role of mTORC2 in regulating gluconeogenesis. Deletion of Sirt1, which positively controls rictor expression and thus mTORC2-mediated Akt Ser473 phosphorylation, causes increased gluconeogenesis in liver (R. H. Wang et al., 2011). The Insulin/Akt/FoxO1 signaling pathway is a major regulator of glucose production and metabolism. Insulinactivated Akt phosphorylates FoxO1, which presents its translocation to the nucleus and leads to its degradation via the ubiquitin proteosome pathway. This causes a decrease in expression of genes involved in gluconeogenesis, such as glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (Hagiwara et al., 2012).
1.2.7.5 Metabolism
In Caenorhabditis elegans, rictor/TORC2 regulates fat metabolism, feeding, growth and life span (Soukas, Kane, Carr, Melo, & Ruvkun, 2009) but little is known about the role of mTORC2 in metabolism in mammalian cells. Rictor-null fibroblasts were reported to display decreased metabolic activity (Shiota, Woo, Lindner, Shelton, & Magnuson, 2006). Colombi and coworkers using genome- wide shRNA screening revealed that mitochondrial dependence increases upon mTORC2 addiction. They identified a group of genes, whose knockdown is selectively lethal in growth factor independent and mTORC2 addicted cells. Several of these genes required for mTOR addiction encode for mitochondrial functions (Colombi et al., 2011).
Page 34
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
1.2.8 mTOR, diseases and the challenges associated with targeting mTOR
Studies confirmed that the mTOR signaling pathway involved in various pathological disorders. The importance of the mTOR pathway in different human diseases including cancer is due to its elevated activity and the biological effects of its downstream target proteins, many of which promote cell survival, proliferation and growth. Therefore, the development of small molecules, which modulate the mTOR pathway, has translational potential into therapy.
Typically, mTOR hyperactivation is caused by inactivating mutations of certain suppressor’s genes in the mTOR signaling pathway like the TSC1/TSC2 complex, LKB1 or PTEN, resulting in mTOR-dependent cell growth (Laplante & Sabatini, 2012).
1.2.8.1 mTOR signalling and cancer
The term cancer designates diseases with uncontrolled cell division resulting in local tumor or neoplasia formation. Cancer cells can also spread to other parts of the body through the blood or lymphatic systems.
Tumor suppressor genes are normal genes that slow down cell division, repair DNA, and eventually induce cells to die (a process known as apoptosis or programmed cell death). When tumor suppressor genes do not work properly, cells may grow out of control, leading to cancer. Many different tumor suppressor genes have been identified, including PTEN, p53, BRCA1, BRCA2, APC, and RB1 (Lai, Visser-Grieve, & Yang, 2012).
Page 35
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Protein Dysfunction Type of cancer
Pro-oncogenes
Upstream of mTOR Gene PI3K Ovarian, gastrointestinal, breast and prostate amplification Gene AKT Ovarian and breast amplification Gene Rheb Breast and head and neck overexpression
Downstream of mTOR
Gene Lung and ovarian, breast, kidney and S6K1 overexpression hepatocellular
4E-BP1 Poor prognostic Breast, colon, ovarian and prostate
Gene elF4E Breast, colon, head and neck overexpression
Tumor suppressor genes
Mutation, PTEN Melanoma, breast, prostate, renal Deletion
LKB1 Mutation Gastrointestinal track
development of Lymphangioleiomyomatosis TSC1/TSC2 Mutation (LAM)
Table 1.1: Pro-oncogenes and tumour suppressor genes linked to the mTOR pathway (Populo, Lopes, & Soares, 2012).
Increased mTOR signaling pathway activity can occur by a number of mechanisms. A common mechanism is the loss of function of the tumor suppressor gene PTEN by mutation, deletion or silencing. The signaling
Page 36
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
pathways that regulate mTOR activity are frequently activated in human cancers (Sawyers, 2003; Wan & Helman, 2007) (Table 1.1). The main mechanism by which mTOR can contribute to cancer development is through its effects on cell cycle progression and its anti-apoptotic activity. mTOR is required for cell cycle progression, and inhibition of mTOR activity by rapamycin arrests cells in the G1 phase of the cell cycle. Expression of a rapamycin- resistant mutant of mTOR reduces the effect of rapamycin on the cell cycle progression.
There is evidence that the effect of rapamycin on the cell cycle progression occurs by the inhibition of the downstream effectors of mTOR, p70S6K, and eIF4E. Moreover, inhibition of mTOR drives the cell into apoptosis. Many proteins in mTOR signaling pathway have already been implicated in cancer (Sawyers, 2003; Wan & Helman, 2007). Therefore, the development of anti- cancer drugs related to the mTOR pathway is conserved to be very promising. Clinical trials have started or conducted using mTOR inhibitors such as rapamycin, RAD001, CCI-799 and AP23579, for cancer treatment.
In clinical trials, rapamycin and three rapamycin analogues, CCI-779 (Temsirolimus), RAD001 (Everolimus), and AP23573 (Deforolimus) have been assessed for their efficacy as anticancer agents (Wan & Helman, 2007). Rapamycin has been tested in clinical studies for the treatment of patients with recurrent PTEN-deficient glioblastoma, phase 1 clinical trial (Cloughesy et al., 2008). Rapamycin analogues were used in clinical trials for the treatment of different cancers type (Table 1.2). Rapamycin and RAD001 can be administered orally. Pharmacokinetic studies showed that both drug have low bioavailability. Therefore, the use of rapamycin as an anticancer might be impractical, because of its poor water solubility and stability in solution. On the other hand, CCI-779 and AP23573 could be administered intravenously. CCI- 779 was approved by U.S Food and Drug Administration (FDA) for treatment of renal cell carcinoma (Morgan, Koreckij, & Corey, 2009).
Page 37
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
More than 50% of ovarian cancers show an activation of at least one out of seven certain signaling pathways (Bast, Hennessy, & Mills, 2009). The most frequent triggered pathway, shared by approximately 70% of ovarian cancers, is the PI3K pathway. Activation of the PI3K pathway can be driven by direct mutation or amplification of genes encoding key members of the pathway like PIK3CA and AKT2, or by inactivating mutations of PTEN (Samuels & Waldman, 2010; Samuels et al., 2004). In many cancers autocrine or paracrine signaling by receptor tyrosine kinases also trigger this pathway (Shaw & Cantley, 2006; Yuan & Cantley, 2008). Akt inhibits the pro-apoptotic Bcl-2 family members Bad and Bax (Cantley, 2002; Katso et al., 2001). It also impedes negative regulation of the transcription factor NF-κB, which leads to an increased transcription of anti-apoptotic and pro-survival genes. By phosphorylation of Mdm2 Akt antagonizes p53-mediated apoptosis. Akt also reduces the production of cell death promoting proteins by down regulation of forkhead transcription factors (Duronio, 2008). In addition, cell proliferation can be stimulated by Akt mediated activation of mTOR (Wendel et al., 2004). mTOR is crucial for the regulation of translation in response to nutrients and growth factors by phosphorylating members of the protein synthesis machinery. This includes the ribosomal protein S6 kinases (p70S6K) and the 4E-binding protein (4E-BP). The latter is responsible for the release of translation initiation factor eIF4E, which is known to have transforming and anti-apoptotic activities in vitro (Schmelzle & Hall, 2000; Wendel et al., 2004). Inhibitors against PI3K and Akt have been shown to inhibit the growth of ovarian cancer xenografts and could potentiate the cytotoxic effects of chemotherapeutics used against ovarian cancer (Hu, Hofmann, Lu, Mills, & Jaffe, 2002; Wen, He, Sun, Li, & Wu, 2014).
Page 38
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
1.2.8.2 Other mTOR-related disorders
The mammalian target of rapamycin (mTOR) acts as a central regulator of ribosome biogenesis, protein synthesis, cell growth, cell survival, cytoskeletal organization and most cell activities.
Dysregulation of mTOR signaling has been reported to be involved in cardiac hypertrophy (Inoki, Corradetti, & Guan, 2005). It also plays an important role in metabolic disorders like obesity, type 2 diabetes, non-alcoholic fatty liver disease and Niemann-Pick type C (NPC) disease (Laplante & Sabatini, 2009; Pacheco & Lieberman, 2008).
An animal study revealed that the mTOR signaling pathway links diet- induced obesity with vascular senescence and cardiovascular diseases (C. Y. Wang et al., 2009). Moreover, it has been shown that inhibition of the mTOR signaling pathway extended the lifespan in invertebrates (yeast, nematodes and fruits) and in mammalians species (mice) (Harrison et al., 2009). Thus, the mTOR signaling pathway appears to be a key target to control many diseases.
1.2.9 mTOR and autophagy
mTOR is a key regulator for cell growth and proliferation, but it also serves as a gatekeeper of cell survival by regulating autophagy and apoptosis. Autophagy is the cellular process in which cells undergo “self-cannibalization” to recycle cellular components and to provide sufficient internal nutrients to maintain cellular homeostasis, especially during starvation(Rabinowitz & White, 2010). Cancer cells maintain high levels of autophagy activity, which is required for tumor growth (Guo et al., 2011; S. Yang et al., 2011). Without growth factors, cells can survive by maintaining energy through autophagy-based catabolism but eventually succumb to death unless growth factor signaling is activated regardless of nutrients availability in the environment (Lum et al., 2005). Similarly, the inhibition of mTOR in the growth factor signaling pathways has
Page 39
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
been linked to autophagy (Blommaart, Luiken, Blommaart, van Woerkom, & Meijer, 1995; Ravikumar et al., 2004), and simultaneous treatment with temsirolimus (mTOR inhibitor) and hydroxychloroquine (autophagy inducer) had synergistic anticancer effect in tumor xenografts by inducing apoptosis (Xie, White, & Mehnert, 2013). The exact molecular mechanism behind how mTOR regulates autophagy was a mystery ever since Blommaart et al. reported rapamycin-induced autophagy in 1995 (Blommaart et al., 1995).
Once again, yeast genetics made a breakthrough for understanding TOR- mediated autophagy regulation in 2000. Ohsumi’s group showed that when yeast cells starved or treated with rapamycin, Atg13 is hypophosphorylated that is associates with Atg1, which in turn leads to kinase activity and autophagy induction (Ohsumi, 2014). In mammalian cells, genetic and biochemistry data demonstrated that under sufficient nutrients, mTOR suppresses autophagy by directly phosphorylating the autophagy initiating kinase ULK1 (UNC-51-like kinase 1, mammalian ortholog of Atg1) on S757, which disrupts the interaction of ULK1 and AMPK (Egan et al., 2011; Kim, Kundu, Viollet, & Guan, 2011). On the other hand, when cells are deprived with glucose, activated AMPK inhibits mTOR and induces autophagy by directly phosphorylating ULK1 at S317 and S777 (Kim et al., 2011).
1.2.10 mTOR and apoptosis
Apoptosis is the balance between pro- and antiapoptotic proteins. Defects in the apoptotic machinery are mainly due to either overexpression of antiapoptotic proteins (i.e. Bcl-2, Bcl-xL and survivin) or decrease of proapoptotic proteins (i.e. BAX and BAK, NBK/Bik, BAD, Par-4, Bim, cytochrome c, apoptosis-inducing factor (AIF)) resulting in uncontrolled growth and proliferation.
Page 40
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Many studies showed that activation of the mTOR signaling pathway is implicated in a decreased expression of proapoptotic proteins. Consistently, mTOR inhibitors induced apoptosis in different cell lines through up regulation of proapoptotic proteins and at the same time down regulation of antiapoptotic proteins (Hayun et al., 2009; Shinjyo et al., 2001; Tirado, Mateo-Lozano, & Notario, 2005).
Eukaryotic initiation factor 4E (eIF4E), downstream of mTOR, is responsible for cap dependent translation and exhibits anti-apoptotic activity (Mamane et al., 2007). Consistently, it has been shown that eIF4E mediates resistance to apoptosis via increases cap-dependent translation (Larsson et al., 2006).
The main biochemical hallmark of apoptosis is activation of caspase-3. Interestingly, caspase-dependent apoptosis is an important mechanism of cell death when the rapamycin derivative, RAD001 is combined with 3 Gyradiations (Albert, Kim, Cao, & Lu, 2006). Additionally, rapamycin can induce caspase-3 activation and induce apoptosis dependent on caspase-3 activation (J. F. Zhang et al., 2006). All these findings indicate that the mTOR signaling pathway is an important target for the development of novel drugs for diseases-associated with apoptosis defect.
1.2.11 mTOR inhibitors
1.2.11.1 First generation mTOR inhibitors: Rapamycin and its analogues
The first mTOR inhibitor to be discovered was rapamycin, a macrolide antibiotic produced by Streptomyces hygroscopicus. Rapamycin strongly inhibits T-cell proliferation and was approved by the FDA in 1999 as an immunosuppressant to prevent organ transplant rejection (Sehgal, 2003). Rapamycin allosterically inhibits mTORC1 through the FRB domain (Figure 1.1) while mTORC2 is insensitive to rapamycin. It has been shown that rapamycin acts by blocking G1 cell cycle progression (Sehgal, 2003). Several studies have
Page 41
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
also shown that rapamycin induces apoptosis through inhibition of hypoxia- induced increases in hypoxia-inducible factor 1α (HIF-1α) (Houghton, 2010). It also induces autophagy and enhances radiation therapy when combined with a Bcl-2 inhibitor that concurrently induces apoptosis (Fumarola, Bonelli, Petronini, & Alfieri, 2014). However, the effects of rapamycin are generally confined to a cytostatic effect and the clinical use of rapamycin as an anti-tumor agent is limited (Guertin & Sabatini, 2009). This therapeutic limitation is thought to be because of its inability to inhibit mTORC2, and the activation of Akt kinase following initial inhibition of mTORC1. Consequences of mTORC1 inhibition results in reducing the its antitumor effects and induction of IRS-1 expression and activation of IGF-I signaling (O'Reilly et al., 2006) (Figure 1.3). Rapamycin treatment induces IRS-1 expression due to inhibition of p70S6K phosphorylation, which results in increased IGF-IR/IRS-1/PI3K signaling to Akt. Reports have shown that p70S6K mediates phosphorylation of IRS-1 inhibitory serine sites (Ser312 and/or Ser636/639) which lead to IRS-1 degradation (B. F. Bahrami et al., 2014; Manning, 2004). Thus, suppression of p70S6K activity by rapamycin may prevent inhibitory IRS-1 phosphorylation, thereby stabilizing IRS-1 (B. F. Bahrami et al., 2014). Currently, rapamycin is only active against certain tumors such as mantle cell lymphoma, renal cell carcinoma and endometrial cancer, in which it is thought to block the effects of mTORC2 by some unexplained mechanism (Q. Liu, Thoreen, Wang, Sabatini, & Gray, 2009).
Owing to poor water solubility, Rapamycin has a low bioavailability. Hence, several analogues of rapamycin with better pharmacokinetic profiles, collectively called “rapalogs”, have been developed. They include RAD001, Temsirolimus and Ridaforolimus (MK-8669) (Figure 1.4); all of these analogues appear to have the same mechanism of action as rapamycin. The US FDA approved Everolimus, originally commercialized as an immunosuppressant, for the treatment of advanced renal carcinoma. Another study has demonstrated that, in addition to mTORC1 inhibition, everolimus inhibits the response of vascular endothelial cells to stimulation by vascular endothelial growth factor Page 42
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
(VEGF) and that its anti-tumor effects are attributed to its anti-angiogenic properties (Houghton, 2010). Temsirolimus is a prodrug of rapamycin and is converted to rapamycin in vivo. Ridaforolimus is still under phase III clinical trials for a variety of cancers that include advanced malignancies such as metastatic soft tissue and bone sarcoma, breast cancer, and relapsed haematological malignancies (Q. Liu et al., 2009).
Rapalogs, like rapamycin, have shown significant activity in clinical trials but their activity is also limited to inhibition of mTORC1. The rapalogs exhibit a cytostatic effect on tumor growth suppression and 18 cancer disease stabilization, rather than regression (Meric-Bernstam & Gonzalez-Angulo, 2009). To circumvent this, they have been combined with other chemotherapeutic agents and better therapeutic efficacies have been observed with combinations over single drug treatment. For instance, the combination of antiestrogen ERA-923 with temsirolimus has been reported for treating breast cancer (Sadler et al., 2006). Rapalogs are also been combined with Insulin-like growth factor I receptor (IGF-IR) inhibitors because of the rapalog-induced Akt activation (feedback loop) observed in cancer cell lines in vitro and in clinical trials (Table 1.2) (O'Reilly et al., 2006).
Page 43
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure1.4: Structures of rapalogs
1.2.11.2 Second generation mTOR inhibitors: Catalytic site (ATP-competitive) inhibitors
Despite the preclinical promise shown by rapalogs, the clinical utility for rapalogs has been limited to a few cancers. Tumor relapse is often observed with rapalogs due to their selectivity for mTORC1 inhibition and the effect of the negative feedback loop on IRS-1. Although they cause a marked hypophosphorylation of p70S6K, rapalogs fail to sustain the dephosphorylation
Page 44
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
and inhibition of 4EBP1, and cell proliferation persists even in the presence of the drug (Fumarola et al., 2014). Interestingly, rapalogs exhibit strong inhibitory activity towards one mTORC1 substrate p70S6K, while inhibition of 4EBP1 is minimal, indicating that it could be an important mechanism of drug resistance. In order to overcome these limitations, novel dual mTORC1/2 inhibitors have been developed and are being studied extensively. These compounds bind to the catalytic domain of mTOR, and inhibit the mTOR kinase activity, thus effectively inhibiting both mTORC1 and mTORC2 (Chresta et al., 2010). The inhibition of mTORC2 is thought to prevent feedback induction of Akt (Hsieh et al., 2012). These ATP competitive inhibitors are highly potent at inhibiting mTORC1 and mTORC2 such that they exhibit inhibitory concentrations (IC50) in vitro in the low nanomolar range as determined by p70S6K phosphorylation at T389 and Akt phosphorylation at Ser473, respectively. They are also very selective such that structurally related kinases are inhibited only at much higher concentrations of drug (Guertin & Sabatini, 2009).
Torin 1 was one of the first dual mTORC1/2 inhibitor developed. It is shown to inhibit proliferation of primary tumor cells to a greater extent than rapamycin (Q. Liu et al., 2009). Shokat and his colleagues have developed PP242 and PP30, both ATP-site mTOR inhibitors exhibiting similar therapeutic profiles to that of Torin1 (Q. Liu et al., 2009). It was assumed that these drugs presented greater therapeutic efficacies than rapalogs because of their ability to inhibit both mTORC1 and mTORC2. Interestingly, it was observed that, in addition to mTORC2 inhibition, the anti-tumor effects of PP242 and PP30 are mediated via more complete inhibition of mTORC1 leading to sustained dephosphorylation and inhibition of 4EBP1 and cap-dependent mRNA translation (Feldman et al., 2009). A number of mTOR catalytic site inhibitors are under preclinical and phase I/II trials and have shown remarkable potency and selectivity for mTORC1 and mTORC2 (Q. Liu et al., 2009). These include KU0063794 (AstraZeneca), INK-128, XL388, OSI-027 and AZD8055. These compounds are under investigation for NSCLC and have demonstrated superior efficacies
Page 45
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
compared to rapamycin and its analogues in preclinical testing (Fumarola et al., 2014; Guertin & Sabatini, 2009; Hsieh et al., 2012; Q. Liu et al., 2009).
AZD8055 is a particularly interesting member of this family of drugs. It is an orally bioavailable, potent, and selective mTOR kinase inhibitor with excellent selectivity for these kinases and modest activity against all class I PI3K isoforms and other members of the PI3K-like kinase family. AZD8055 has shown proven efficacy in NSCLC cell lines superior to that of rapamycin (Guertin & Sabatini, 2009). AZD8055 has been shown to significantly decrease the phosphorylation of 4EBP1 on the rapamycin-insensitive T37/46 sites and to potently inhibit cap- dependent translation at low nanomolar concentrations with concomitant inhibition of cell proliferation. It is also thought to promote a greater autophagosome formation and autophagy activation than rapamycin. In addition, AZD8055 induced significant growth inhibition and regression in NSCLC xenografts (Fumarola et al., 2014). Similar to the rapalogs, catalytic site mTOR inhibitors have also been combined with inhibitors of the PI3K-Akt pathway to overcome the negative feedback of mTORC1 inhibition on PI3K/Akt and MAPK signaling (Q. Liu et al., 2009). AZD8055, in combination with MEK 1/2 inhibitor AZD6244 (selumetinib) has demonstrated superior anti-tumor efficacy over either drug given as single agents (Fumarola et al., 2014). Some ATP- competitive inhibitors show dual activity against mTORC1/2 as well as activity against the p110 subunit of PI3K due to the structural similarity of the catalytic domains of these proteins, and are called dual mTOR-PI3K inhibitors. These agents have the potential of completely shutting down the PI3K/Akt/mTOR pathway but it may cause greater toxicity. Examples of this class of drugs are NVPBEZ235 and XL-765 (SAR245409); these compounds are currently under phase I/II clinical trials for NSCLC and breast cancer (Table 1.2) (Fumarola et al., 2014; Q. Liu et al., 2009).
Page 46
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
mTOR Mechanism of action inhibitors Rapamycin and analogues Binding to the immunophilin FKBP12 Deforolimus Partial mTORC1 inhibitor Cell-type specific mTORC2 inhibitor Binding to the immunophilin FKBP12 Everolimus Partial mTORC1 inhibitor Cell-type specific mTORC2 inhibitor Binding to the immunophilin FKBP12 Sirolimus Partial mTORC1 inhibitor Cell-type specific mTORC2 inhibitor Binding to the immunophilin FKBP12 Temsirolimus Partial mTORC1 inhibitor Cell-type specific mTORC2 inhibitor Small molecule inhibitors of kinases AZD8055 ATP competitive inhibitor of mTOR Ku-0063794 Specific mTORC1 and mTORC2 inhibitor PP242 mTOR kinase inhibitor PP30 mTOR kinase inhibitor Torin1 mTOR kinase inhibitor WYE-354 ATP competitive inhibitor of mTOR mTOR and PI3K dual-specificity inhibitors NVP-BEZ235 ATP-competitive inhibitor of PI3K and mTOR PI-103 ATP competitive inhibitor of DNA-PK, PI3K and mTOR PKI-179, PKI-587 ATP competitive inhibitor of DNA-PK, PI3K and mTOR XL765 ATP-competitive inhibitor of DNA-PK, PI3K and mTOR
Table 1.2: mTOR inhibitors in clinical trial
Page 47
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
1.3 Autophagy
Autophagy is a catabolic process by which cells degrade intracellular components in lysosomes. It includes three different variants: macroautophagy, microautophagy, and chaperone-mediated autophagy. Among them, macroautophagy (hereafter referred to as autophagy) is the most extensively studied form, which engulfs intracellular constituents into double membrane vesicles, termed autophagosomes, which then fuse with lysosomes to form autolysosomes, where the autophagic contents are degraded. Distinct from proteasomal degradation, which only proteolyse soluble proteins inside the proteasomal barrels, autophagy is the only cellular mechanism for degrading large cellular components such as protein aggregates and entire organelles. The substrates of autophagy may include cytoplasm, organelles, proteins and protein aggregates as well as the autophagic components that associated with the inner membranes of autophagosomes (Figure 1.5).
The cellular garbage disposal and intracellular recycling provided by autophagy serves to maintain cellular homeostasis by eliminating superfluous or damaged proteins and organelles, and invading microbes, or to provide substrates for energy generation or biosynthesis in stress. Thus, autophagy promotes the health of cells and animals and is critical for development, differentiation, maintenance of cell integrity and function as well as for the host defence against pathogens. Deregulation of autophagy is linked to susceptibility to various disorders including degenerative diseases, metabolic syndrome, aging, infectious diseases and cancer. Given that cancer is a complex process and autophagy exerts its effects in multiple ways, role of autophagy in tumorigenesis is context-dependent. Our understanding of the molecular mechanism and regulatory pathways of autophagy has substantially improved by studies of numerous investigators over the past thirty years. The next challenge will be to understand the physiological significance of autophagy in various cellular contexts as well as how to modulate autophagy for therapeutic purposes in these settings (White, 2014).
Page 48
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
1.3.1 Molecular machinery of autophagy
The central machinery of autophagy includes a series of complexes comprised of autophagy-related (Atg) proteins, executing the formation of autophagosomes: membrane nucleation, expansion, closure and maturation by fusing with lysosomes (Figure 1.5). In mammals, the Atg1/Ulk1 complex (Unc51-like kinase 1; the mammalian homolog of yeast Atg1) consisting of kinase Ulk1, two scaffold proteins Atg13 and FIP200 (focal adhesion kinase family interacting protein of 200-kDa) and Atg101 receives signals from mTORC1 (mammalian target of rapamycin complex 1), an integrator of growth factors, nutrients and energy status (Mercer, Kaliappan, & Dennis, 2009; Z. Yang & Klionsky, 2010). During nutrient replete conditions, mTORC1 phosphorylates Ulk1 and Atg13 and negatively regulates Ulk1 complex. Upon starvation, dissociation of mTORC1 followed by a series of dephosphorylation/phosphorylation events activates the Ulk1 complex (Hosokawa et al., 2009; Jung et al., 2009). Although the direct targets of Ulk1 complex have not been identified, current evidence supports its roles in activation of Beclin1/Vps34 (Bcl-2 interacting coiled-coil protein 1/ vacuolar protein sorting 34) complex and thus contributes to autophagosome induction as well as maturation (Neufeld, 2010).
Several Beclin1-containing complexes that dictate the sequential process of autophagosome formation have been identified. The core components include the membrane anchor p150, Beclin1, PI3KC3 and Vps34, which generates phosphatidylinositol 3-phosphate for protein recruitment. In addition, Atg14L (Atg14-like; the mammalian homolog of yeast Atg14) and UVRAG (UV irradiation resistance-associated gene) interact with Beclin1 in a mutually exclusive fashion. The Beclin1/Vps34/Atg14L complex is involved in autophagosome initiation. UVRAG binds Bif-1 (Bax-interacting factor 1; endophilin B1), which deforms membrane and is involved in membrane elongation. The UVRAG complex also promotes the fusion of autophagosomes with lysosomes. As a negative regulator, Rubicon (RUN domain and cysteine-
Page 49
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
rich domain containing, Beclin1-interacting) inhibits the function of UVRAG complex in autophagosome maturation (Matsunaga et al., 2009; Zhong et al., 2009).
Two ubiquitin-like conjugation systems act in expansion and closure of autophagosome membranes. The ubiqutin-like protein Atg12 is linked to Atg5 by the E1-like activating enzyme Atg7 and E2-like conjugating enzyme Atg10. Atg12-Atg5 then associates with Atg16L (Atg16-like) to form a multimeric complex. In the second conjugation reaction, another ubiqutin-like molecule LC3 (microtubule-associated protein 1 light chain 3) is processed by the cysteine protease Atg4 to reveal a C-terminal glycine (LC3 I). LC3 I is cleaved and linked onto phosphatidylethanolamine (PE) by the E1-like Atg7 and the E2- like Atg3, and translocates from cytoplasm to autophagosomal membrane (LC3 II). The translocation of LC3 is used to monitor autophagy induction and the ratio of protein levels of LC3 II to LC3 I is used as an indication of autophagy levels. Atg4 also cleaves the lipidated LC3 II and frees it from the membrane (Kirisako et al., 2000; Satoo et al., 2009).
Conversely, the LC3 lipidation system is required for the formation of Atg16L complex (Fujita et al., 2008; Sou et al., 2008), indicating interconnection of these two systems (Z. Yang & Klionsky, 2010).
Autophagy can function in the non-selective bulk degradation of cytoplasmic contents or in the selective turnover and elimination of specific cellular components. To selectively target substrates to autophagosomes, protein and organelle substrates are conjugated with ubiquitin, while selective autophagic degradation of lipid droplets may use other signals (Komatsu et al., 2007; Oku & Sakai, 2010). Autophagy cargo receptors such as p62/SQSTM1 and NBR1 (neighbour of BRAC1 gene1), interact with both the ubiquitin on cargo and with the autophagy machinery through LC3-interaction (LIR) domains. This enables the cargo receptors to recognize and deliver cargo to autophagosomes (Ichimura, Kominami, Tanaka, & Komatsu, 2008; Kirkin, Lamark, Johansen, & Page 50
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Dikic, 2009). Cargo receptors are degraded along with the cargo and their accumulation, often in large aggregates in the case of p62/SQSTM1, is symptomatic of autophagy inhibition (Komatsu et al., 2007) (Figure 1.5).
Page 51
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
l.QI • cC Soluble CO'Yl)OI...O Pl'oCW\~
c-,...... ""' ---'---
Figure1.5: Machinery and regulators of autophagy The process of autophagy, from autophagosome formation—nucleation, expansion, to maturation—are presented along with molecular machinery that regulates it.. The major negative regulator of autophagy, mTORC1, which Page 52
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
combines modulators including availability of nutrients or growth factors, energy depletion, or hypoxia, is also shown. Drug target proteins are highlighted with striated outlines. Autophagy stimulators are indicated by green boxes, autophagy inhibitors by red boxes. Question marks represent inhibitors aiming at potential targets, the kinase Ulk1, the cysteine protease Atg4, and the E1-like ubiquitination enzyme Atg7. PE, phosphatidylethanolamine; IP3, inositol-1,4,5- triphosphate; IP3R, IP3 receptor. Adapted from: (H. Y. Chen & White, 2011).
1.3.2 Regulation of autophagy
The availability of nutrients, growth factors, and hormones as well as stress, regulates autophagy. mTORC1 is a major negative regulator of autophagy. mTORC1 promotes protein synthesis, cell division and metabolism in response to the nutrient, growth factor and hormone availability, while suppressing autophagy. Growth factors such as insulin or insulin-like growth factors (IGFs) activate signal cascades Ras-Raf-MEK (MAP kinase/ERK kinase)-ERK1/2 and PI3K-Akt. Both signal pathways suppress the upstream repressor of mTORC1, TSC1/2, a GTPase-activating protein complex of the mTORC1 activator Rheb (Ras homolog enriched in brain), thereby keeping mTOCR1 active and autophagy suppressed in growth factor condition. Tumor cells commonly take advantage of the growth-promoting function of mTOR by acquiring activating mutations in PI3K or inactivating mutations in the PI3K signaling antagonist PTEN (Guertin & Sabatini, 2009); therefore, suppression of autophagy may constantly occur in some tumors. Dependent on the availability of amino acids, mTORC1 is targeted to lysosomal membranes by the Regulator (Ras-related GTPase) complex, which is essential for mTORC1 activation (Sancak et al., 2010). The internal energy status of cells impinges on AMPK, which activates autophagy by inhibiting mTORC1 activity when energy is depleted. Stress such as hypoxia actives AMPK and TSC1/2 complex and induces autophagy. Hypoxia also exerts its effect on autophagy through the hypoxia-inducible factors (HIFs)-mediated transcription of Bnip3, which interacts with and inhibits
Page 53
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Rheb (Efeyan & Sabatini, 2010; Neufeld, 2010) (Figure 1.5). Thus, through inhibition of autophagy, mTORC1 coordinates cell growth and catabolism in response to extracellular stimuli. Autophagy is also regulated by mTORC1- independent signaling. Lowering inositol or IP3 induces autophagy. The physiological relevance that IP3 regulates autophagy is still unclear. IP3 receptor was shown to bind Beclin1, which suppresses autophagy (Vicencio et al., 2009). Nevertheless, how IP3 exactly suppresses autophagy remains elusive. In skeletal muscle, Akt represses autophagy independent mTORC1 by phosphorylating and inhibiting FOXO (forkhead box O) transcription factors, which control the transcription of atg genes (Mammucari et al., 2007), likely providing a tissue specific mechanism of autophagy regulation. Hypoxia also induces autophagy in an mTOR independent manner through HIFs-mediated transcription by inducing Bnip3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3) to relieve the inhibition of Bcl-2 on Beclin1 (Mazure & Pouyssegur, 2010).
1.3.3 Role of autophagy in physiology
1.3.3.1 Autophagy-mediated protein quality control
Autophagy constitutively degrades excess or damaged proteins and organelles through its basal activity, which is critical to maintain cellular homeostasis and function. This is especially important for post-mitotic cells, which cannot dilute cellular waste products through cell division. Impaired autophagy in mice causes quiescent cells such as neurons and hepatocytes, to accumulate ubiquitin- and p62/SQSTM1-positive protein inclusions, aberrant membranous structures and deformed mitochondria, accompanied by neuronal degeneration and liver injury (Hara et al., 2006).
Page 54
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
1.3.3.2 Autophagy-mediated organelle quality control
Autophagy eliminates damaged organelles and ensures their quality control, thereby maintaining organelle function and preventing the harmful consequences of organelle damage. Autophagy may sustain cellular metabolism through mitochondrial quality control, while impaired autophagy may lead to compromised or altered metabolism in part through the accumulation of dysfunctional mitochondria (Mizushima & Levine, 2010).
1.3.3.3 Autophagy in cellular remodelling and development
Reports indicate that autophagy plays a significant role in cellular remodelling and contributes to cell differentiation and development. How autophagy exactly facilitates the cellular rearrangement process, remains unclear. Autophagy- mediated non-selective bulk degradation and selective elimination of specific cellular components such as organelles, structural proteins and regulatory factors may both involve in this process. During development, the autophagic protein Atg5 is required for mouse embryos to develop beyond the four-cell to eight-cell stages (Tsukamoto et al., 2008). Role of autophagy in this preimplantation development is still unknown.
Autophagy is also important for differentiation. In some cases, this activity described as the autophagic elimination of mitochondria or mitophagy (Twig, Hyde, & Shirihai, 2008).
During adipogenesis, autophagy is necessary for lipid accumulation and formation of a single large lipid droplet in white adipocytes. Autophagy-deficient white adipocytes contain multilocular lipid droplets and increased mitochondria (Singh et al., 2009). It remains to investigate whether defective mitochondrial clearance is a cause of the impaired differentiation of white adipocytes.
Page 55
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Young et al reported that autophagy mediating the acquisition of the oncogene-induced senescence phenotype (Young et al., 2009). When senescence occurs, cells undergo substantial remodelling and acquire a phenotype of stable cell cycle arrest but remain metabolically active.
1.3.3.4 Autophagy in metabolic homeostasis
Recent findings have uncovered multiple roles of autophagy in lipid homeostasis, defects in which can lead to metabolic disorders and fatty liver disease. Fatty liver ranges from simple steatosis to steatohepatitis, cirrhosis and ultimately can lead to HCC (H. Y. Chen & White, 2011).
It is suggested that dysregulation of lipid metabolism or hepatic lipotoxicity could be a trigger of inflammation and fibrogenesis, which are associated with the development of aggressive disease (Trauner, Arrese, & Wagner, 2010).
Although it is still unclear how exactly autophagy deficiency contributes to initiation or progression of disease, failure of lipid homeostasis leading to lipotoxicity and chronic inflammation, in addition to p62/SQSTM1 accumulation, is a likely possibility.
Autophagy is important for accessing lipid stores through lipophagy and promoting lipid breakdown and preventing deposition in liver (Singh et al., 2009). Unfortunately, factors that disrupt lipid homeostasis commonly suppress autophagy, leading to a vicious cycle.
Autophagy declines with age, correlating with altered metabolism manifested as ectopic fat deposition and intracellular garbage accumulation (Levine & Kroemer, 2008). This suggests that the imbalance of lipid homeostasis as well as waste accumulation and cellular functional degeneration due to suppressed
Page 56
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
autophagy may accelerate aging. In worms, autophagy is required for lifespan extension provided by dietary restriction (Levine & Kroemer, 2008).
1.3.3.5 Autophagy confronts stress and environmental insults
Autophagy is upregulated in response to stress, including growth factor and nutrient limitation, energy depletion and hypoxia. In yeast, starvation induces autophagy, which recycles intracellular constituents to support metabolism, leading to adaptation and survival. In mammals, this self-cannibalistic function is conserved, and autophagy deficient mice cannot survive the neonatal starvation period and show indications of energy depletion (Kuma et al., 2004).
The capability of autophagy to degrade proteins, lipids, glycogen and nucleic acids provides cells the flexibility to utilize intracellular components or various stores of nutrients for energy production and biosynthesis under stress (Kuma & Mizushima, 2010; Rabinowitz & White, 2010). Autophagy generates substrates such as nucleosides, amino acids, fatty acids and sugars from the breakdown of intracellular components.
Metabolism of different substrates can produce unequal redox equivalents such as NADPH, which can support lipid biosynthesis and maintain cytosolic redox equilibrium (Noguchi et al., 2009; Vander Heiden, Cantley, & Thompson, 2009). Therefore, through selective autophagy, different substrates may support central metabolism by different substrates to restore metabolic homeostasis in redox status and in biosynthesis in addition to in energy and leading to adaptation.
Page 57
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure1.6: Regulation and functions of the autophagic pathway A range of stresses and stimuli regulates autophagy. This has diverse effects on the cell, achieving a number of functions, both through autophagic degradation of specific elements themselves, or by interaction with other pathways such as apoptosis. ER: endoplasmic reticulum. Adapted from (King, 2012).
1.3.3.6 The importance of controlling p62/SQSTM1 levels by autophagy
The capability of autophagy to eliminate specific proteins selectively has an important role in cellular function. The autophagic substrate receptor p62/SQSTM1, which is induced by stress to facilitate selective autophagic
Page 58
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
degradation, is a substrate of autophagy (Bjorkoy et al., 2005; Ichimura, Kumanomidou, et al., 2008). p62/SQSTM1 contains oligomerization and protein interaction domains and facilitates protein aggregate formation (Komatsu et al., 2007; Moscat, Diaz-Meco, & Wooten, 2007) (Figure 1.5). Failure to clear p62/SQSTM1 due to impaired autophagy causes liver damage in mice and promotes tumorigenesis of allografts (Komatsu et al., 2007). Thus, autophagic degradation of a specific protein, p62/SQSTM1, has a role in preventing disease.
p62/SQSTM1 is required for oncogenic Ras-driven NF-κB activation and lung adenocarcinoma in mice (Duran et al., 2008). By contrast, aberrant p62/SQSTM1 accumulation in autophagy-defective cells abrogates NF-κB signaling that may promote tumorigenesis in the liver (Mathew et al., 2009). The role of NF-κB in tumorigenesis in the liver is becoming clear. NF-κB activates prosurvival and proinflammatory gene transcription. Therefore, role of NF-κB in tumorigenesis is context dependent. It will be interesting to delineate the interplay between autophagy deficiency-dependent p62/SQSTM1 accumulation and NF-κB signaling in tumorigenesis of different tissue types.
Furthermore, p62/SQSTM1 accumulation activates Nrf2-mediated transcription. Studies indicate that the autophagy deficiency-dependent p62/SQSTM1 accumulation alters the regulation of another signaling pathway, transcription by Nrf2 (NF-E2 related factor 2). Nrf2 is a transcription factor mediating transcription of antioxidant and detoxifying genes. p62/SQSTM1 interacts with Keap1 (kelch-like ECH-associated protein 1), an adaptor for the cullin-based E3-ligase that promotes Nrf2 degradation. By sequestrating Keap1, p62/SQSTM1 accumulation causes activation of the Nrf2-targeted genes (Jain et al., 2010; Lau et al., 2010). It will be of interest to see if the autophagy- dependent accumulation p62/SQSTM1 promotes tumorigenesis through activating Nrf2.
Page 59
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
1.3.4 Autophagy and tumorigenesis
Tumorigenesis is a complex multistage process. It includes tumor initiation, promotion, and progression to malignancy and metastasis. This process involves profound alteration of cells in terms of growth, proliferation, metabolism, stress tolerance and survival, and interaction with the microenvironment where they grow. Genetic and epigenetic changes initiate and facilitate progression of normal cells toward malignancy, and chronic tissue damage provides a pro-mutagenic environment to accelerate this process. Chronic inflammation creates a cancer-promoting environment to support survival and proliferation of abnormal cells and hastens progression.
1.3.4.1 Autophagy suppresses tumor initiation by limiting genome mutation
The housekeeping function of autophagy maintains turnover of proteins and organelles and ensures homeostasis and cellular health, preventing disease conditions. In response to stress, autophagy eliminates damaged proteins and organelles. This damage mitigation of autophagy can be important for survival of tumor cells, which are subjected commonly to metabolic stress due to insufficient vascularization. Autophagy-deficient murine cells are although more susceptible to metabolic stress. These cells have evident DNA damage response activation and have an increased frequency of chromosome gains and losses (Mathew et al., 2007). Autophagy deficient mice are tumor-prone and liver tumors there from, as well as human liver tumors accumulate p62/SQSTM1-containing protein aggregates, ER chaperones and activate the DNA damage response (Mathew et al., 2009). Thus, by taking out the garbage, autophagy may suppress genomic instability to limit tumor initiation and progression.
Autophagy may also hinder proliferation of cells with cancer mutations, in addition to limiting genomic mutations in cells. Inhibition of autophagy delays
Page 60
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
oncogene-induced senescences (Young et al., 2009). It is believed that senescence is a tumor suppressive mechanism attributed to the lack of proliferation of senescent cells. By facilitating senescence, autophagy limits propagation of oncogenic mutations thereby suppressing tumorigenesis.
1.3.4.2 Autophagy suppresses tumor initiation and progression by limiting chronic inflammation
Autophagy maintains homeostasis by removing excess or damaged intracellular components and microbial invaders, and by regulating lipid metabolism. This not only restrains damage, including genome instability, but also the subsequent inflammation. In autophagy suppressive conditions, persistence of unresolved damage leads to chronic cell death and inflammation. In liver, and probably other tissues, this can provide a cancer-promoting environment (H. Y. Chen & White, 2011).
Autophagy provides an internal resource to support metabolism and mitigates damage, allowing cells to survive stress. Autophagy-deficient murine tumor cells in a background of defective apoptosis, which commonly occurs in tumors, undergo necrosis when subjected to metabolic stress (Degenhardt & White, 2006). Therefore, autophagy limits genetic instability and inflammation that predisposes to tumor initiation, promotion and progression. In this regard, autophagy stimulation may be tumor preventive.
1.3.4.3 Tumor cells with oncogenic mutations may be more dependent on autophagy for survival
Tumor cells may have differing requirements for autophagy, dependent on the transformation stages and tumor types. With respect to metabolism, autophagy may promote tumor progression by supporting survival and proliferation of cancer cells especially those of an aggressive tumor that has
Page 61
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
increased metabolic demand. During cancer progression, cells often activate pathways that lead to changes in metabolism, such as altered or increased nutrient requirements for energy production and biosynthesis. For example, oncogenic Myc causes cells to become addicted to glutamine for energy production (Wise et al., 2008). Activation of oncogenic pathways may also increase metabolic demand due to promotion of cell proliferation and growth. While autophagy is a pathway that helps to confront these extreme situations, cancer cells may require it to meet their particular metabolic demands. In this regard, inhibition of autophagy may suppress tumor progression.
1.3.4.4 Autophagy inhibition sensitizes tumor cells to cell death
Since autophagy is a stress survival pathway, aggressive cancer cells may become more sensitive to autophagy inhibition. The autophagy inhibitor chloroquine (CQ), which induces lysosomal stress and prevents autophagic degradation, preferentially kills Myc-expressing mouse cells in vitro in a p53- dependent manner. CQ impairs spontaneous lymphomagenesis in Myc- transgenic mice that model Burkitt’s lymphoma and in atm-deficient mice that model ataxia telangiectasia also dependent on p53 (Maclean, Dorsey, Cleveland, & Kastan, 2008). Thus, lysosomal stress and/or autophagy inhibition may serve as a stress to enhance tumor suppression pathways to harness tumorigenesis when cancer mutations exist.
In a p53-deficient, Myc-induced mouse model of lymphoma, inhibition of autophagy promotes cell death and delays the recurrence of tumors following p53 reactivation or alkylating agent treatment (Amaravadi et al., 2007). This suggests that once cancer occurs, tumor cells may rely on the cytoprotective function of autophagy to survive tumor suppressor reactivation or therapeutic stress that induces cell death and to survive the tumor microenvironment during latency (White & DiPaola, 2009)
Page 62
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
1.4 Monepantel
1.4.1 History
Parasitic infections by nematodes represent a serious threat to the health of humans, companion animals and livestock. Three classes of broad-spectrum anthelmintics have been available over the past three to four decades to control these parasites. Unfortunately, some of these anthelmintics have, over time, decreased in effectiveness with the appearance of resistant parasite populations (Waller, Rudby-Martin, Ljungstrom, & Rydzik, 2004). As a result, the development of compounds from new drug classes that are able to control such resistant strains became essential. The recently identified class of AAD anthelmintics offers an attractive solution, either used alone or alternating with older anthelmintic classes. Monepantel (MPL) is a novel anthelmintic drug from AADs, introduced in the late 2000's. Due to its favourable pharmacological profile, it is the first representative of the amino acetonitrile derivatives, which is the first chemical class with a new mode of action against livestock nematodes introduced after ivermectin in the 1980s. It took almost 30 years to discover and introduce an active ingredient with a new mode of action: ivermectin was introduced in 1981 and monepantel in 2009 in New Zealand (ZOLVIX) (Kaminsky et al., 2008). It has been shown to be efficacious against various species of livestock-pathogenic nematodes (Kaminsky, Mosimann, Sager, Stein, & Hosking, 2009), and has thus been approved for certain veterinary uses (Kaminsky et al., 2008; Rufener, Keiser, Kaminsky, Maser, & Nilsson, 2010). Since 2009, monepantel has been introduced as a drench for sheep in several countries, e.g. New Zealand, EU, Australia, Uruguay, Argentina, etc. It is approved for use on goats in some countries. So far, there are no monepantel products for cattle and it is not used on dogs and cats. Furthermore, it has been shown to be ineffective in human soil-transmitted helminthiasis hence hindering its development as a human anthelmintic (Tritten, Silbereisen, & Keiser, 2011).
Page 63
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
1.4.2 Chemical structure
Monepantel [N-[(1S)-1-Cyano-2-(5-cyano-2-trifluoromethyl-phenoxy)-1- methylethyl]-4- trifluoromethylsulfanylbenzamide (C20H13F6N3O2S) (CAS NO 887148-69-8)] consists of two phenyl rings connected by a short chain structure (Figure 1.7). It exists in two enantiomeric forms with only the S-enantiomer being biologically active (Ducray et al., 2008). It only differs from other members of AADs by the chemical substituents on the phenyl rings and can be chemically synthesized through alkylation of phenols with chloroacetone, Strecker reaction and acylation of the amine with aryl chlorides (Kaminsky et al., 2008).
Figure1.7: Chemical structure of monepantel (MPL)
Page 64
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
1.4.3 Physicochemical properties
Under room temperature (below 30⁰C), the compound is stable for its entire shelf life in fluorinated High Density Polyethylene (HDPE) bottle or laminated aluminium pouches and it will remain stable for 12 months after initial opening. MPL also has very low water solubility (0.08mg/L) but is soluble in lipid and ethanol [APVMA (2010) Public Release Summary on the Evaluation of the New Active Monepantel in the Product Zolvix Monepantel Broad Spectrum Oral Anthelmintic for Sheep. Australian Pesticides and Veterinary Medicines Authority (APVMA), pp. 1-79].
1.4.4 Mode of action
In susceptible nematodes, similar to most other anthelmintics with the exception of benzimidazoles (Borgers & De Nollin, 1975; R. J. Martin, 1997), MPL affect ligand-gated ion channels leading to interference of signal transduction, possibly at their neuromuscular synapses (Kaminsky et al., 2008; Rufener, Baur, Kaminsky, Maser, & Sigel, 2010). The affected parasites then experience deregulation in muscle contraction, paralysis, necrosis and finally expulsion from the host. So far, three nicotinic acetylcholine receptors (nAChR) related genes have identified as the primary targets of MPL (Kaminsky et al., 2008). Interestingly, all of the three genes encode for the proteins representing the DEG-3 subfamily of nAChR subunits that are only present in nematodes, which may well explain the exceptional safety of MPL in mammals (Mongan, Jones, Smith, Sansom, & Sattelle, 2002; Rufener, Keiser, et al., 2010). MPL able to modulate DEG-3/DES-2 and ACR-23 channels, encoding nAChR α- subunit with resemblance to that of α7 subunit in second transmembrane domain (Rufener, Baur, et al., 2010). Studies on C. elegans indicate that the DEG-3 and DES-2 gene, while being expressed simultaneously, are capable to form a choline-activated ion channel, which is known to be partially permeable to Ca2+ (Treinin, Gillo, Liebman, & Chalfie, 1998; Yassin et al., 2001). The activity of DEG-3/DES-2 can be enhanced by endoplasmic reticulum-resident
Page 65
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
chaperonin RIC-3. In fact, RIC-3 (Resistant to Inhibitors of Cholineserase-3) also plays a role in the DEG-3/DES-2 receptor function by inducing fast and almost complete desensitization (Ben-Ami et al., 2005; Halevi et al., 2002). Even though MPL does not activate the DEG-3/DES-2 channels, it is able to potentiate the late currents after stimulation of the channels by its preferred substrate, choline (Rufener, Baur, et al., 2010).
MPL is not the only anthelmintic compound that affects nAChR since other anti-parasitic drugs such as levamisole; pyrantel and morantel also known to be nAChR agonists albeit of different classes. Levamisole and related compounds activate N-, L- and B- nAChR subtypes (R. J. Martin, 1997). A comprehensive study on AADs showed that there is no cross resistance between AADs and these drugs, thus suggesting MPL acts through a novel mechanism (Kaminsky et al., 2008).
1.4.5 Pharmacokinetics of monepantel
The plasma disposition kinetics of MPL has been assessed in sheep after its intravenous and oral administration (Karadzovska et al., 2009). Monepantel sulphone was the main metabolite detected in the bloodstream after MPL administration. As this metabolite is also active against nematodes, the pharmacokinetic behavior of MPLSO2 is relevant for the interpretation of residue and efficacy studies (Karadzovska et al., 2009). Although the evaluation of drug concentration profiles in the bloodstream contributed with useful information,
MPL and MPLSO2 exert their anthelmintic effects in some non-vascular target tissues such as the gastrointestinal (GI) tract, where nematode parasites are located (Karadzovska et al., 2009).
In the orally treated sheep, MPL exhibited a higher Cmax and greater AUC than in the sheep with intra-ruminal and intra-abomasal way of MLP administration (Hosking et al., 2009). After oral administration, monepantel is
Page 66
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
quickly absorbed into the blood stream and rapidly metabolized to monepantel sulfone derivative that has a similar efficacy as the parent molecule. Four hours after administration, the sulfone derivative predominates over the parent molecule. Mean residence time in blood for monepantel and monepantel sulfone are about 5 and 110 hours, respectively (Karadzovska et al., 2009). Similar results were observed for the MPL.SO2: sheep treated orally had greater values of all pharmacokinetic parameters. The higher plasma concentrations of MPL and MPL.SO2 observed in the orally treated lambs compared with the lambs treated by intra-ruminal or intra-abomasal administration may be the basis for the better efficacy of oral administered MPL, and hence a reason why oral treatment with a suitable drenching technique is recommended (Hosking, Kaminsky, Sager, Rolfe, & Seewald, 2010).
About 95% of the administered dose is immediately metabolized to the sulfone derivative. The maximum blood concentration of the sulfone derivative is reached about 2 hours after administration. The recent work corroborated that
MPL and MPLSO2 plasma concentrations were significantly lower than those measured in the GI tract (Lifschitz et al., 2014).
Concerning MPL elimination, a rather small fraction of the parent drug (3·7%) is excreted via faeces and the remaining drug is mainly converted to the sulfone metabolite. Besides sulfoxidation to MPL.SO2, parent drug is also metabolized via other pathways and eliminated as other MPL metabolites (Stuchlikova et al., 2014).
1.4.6 Toxicology
Acute toxicity studies demonstrated that MPL has low acute oral and dermal toxicity in rats (LD50s both >2000 mg/kg bodyweight (bw)). Other safety pharmacological studies in rats indicate that MPL does not exert negative effects at a dose of 2000 mg/kg bw (APVMA, 2010). The recommended dose of
Page 67
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
MPL is 2·5 mg/kg bw in sheep and 3·75 mg/kg bw in goats. This dose is effective against all major gastrointestinal nematodes, including those that are resistant to benzimidazoles, imidazothiazoles and macrocyclic lactones, and a commercialized combination of derquantel and abamectin (Kaminsky et al., 2011).
1.5 Aim of the study and hypothesis
The realization that MPL is a very well tolerated agent in mammals coupled with the fact that the other major antiparasitic treatments such as the macrocyclic lactones, imidazothiazoles and in particular the benzimidazole carbamates have all shown some degree of anticancer activity encouraged us to evaluate MPL in laboratory based anticancer tests. In our previus studies on benzimidazole carbamates (BZD), results indicated on anticancer properties for this group of agents and in particular for albendazole in ovarian cancer (Pourgholami et al., 2010; Pourgholami, Yan Cai, Lu, Wang, & Morris, 2006). Towle et al and Naito et al in their separate investigations demonstrated anticancer activity of macrocyclic lactones and imidazothiazoles in pre-clinical models of cancer (Naito et al., 1998; Towle et al., 2001).
While there was no previus study with MPL on human and cancer and with respect to previus finidings, we therefore put MPL in a progressive series of tests starting from visualization of its microscopic effects on cells, to cellular and molecular. This research aims to determine the efficacy and selectivity of MPL and to elucidate its mechanism of action using in vitro and in vivo models of ovarian cancer cell lines. Our working hypothesis is that MPL targets the mTOR / p70S6K pathway to induce autophagy along with cell cycle arrest and antiproliferative effects with minimal effect on noncancerous cells or tissues.
Page 68
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
This hypothesis will be tested through the following objectives:
- Evaluate the efficacy and selectivity of MPL on cultured different ovarian cancer cell lines Vs. non-cancerous models - Determine the mechanism of action and anti-tumor efficacy of MPL in ovarian cancer using in vitro and in vivo models
Results obtained through this study will identify the molecular effects of treatment with MPL on ovarian cancer cells, and provide rationale for pri- clinical development and application of MPL and progression to clinical trials.
Page 69
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Chapter 2: Material and Methods
2.1 Material Novartis kindly gifted Monepantel (MPL), Basel, Switzerland or MPL purchased from contract manufacturer. Unless otherwise stated, all other chemicals or reagents used in this study were purchased from Sigma-Aldrich (Sydney, Australia).
The following primary antibodies used throughout this study:
Molecular Antibody Cat. No. Source Manufacturer Dilution Weight (kDa) 4E- 9644S 15-20 Rabbit Cell Signalling 1/1000 BP1(49D7) P-4E-BP1 (Thr37/46) 2855S 15-20 Rabbit Cell Signalling 1/1000 (236B4) 1/35,000- anti-GAPDH G9545 38 Mouse Sigma 1/70,000 anti mouse 1/500 – IgG HRP- sc-2302 Goat Santa cruz 1/5000 linked anti-rabbit IgG, HRP- 7074 Cell Signalling 1/1000 linked Beclin-1 3738 60 Rabbit Cell Signalling 1/1000 32-35 Caspase-3 9662S Rabbit Cell Signalling 1/1000 17-20 Caspase-8 4790S 18, 57 Rabbit Cell Signalling 1/1000 1/500 – CDK2 (H298) sc-748 34 Rabbit Santa Cruz 1/5000 1/500 – CDK4 (C22) sc-260 34 Rabbit Santa Cruz 1/5000 Cyclin A 4656 55 Mouse Cell signalling 1/1000 (BF683)
Page 70
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
cyclin D1 2926 36 Mouse Cell Signalling 1/1000 (DCS6) cyclin E2 4132 48 Rabbit Cell Signalling 1/1000 LC3B 2775S 14, 16 Rabbit Cell Signalling 1/1000 mTOR 2983S 289 Rabbit Cell Signalling 1/1000 (7C10) P-mTOR 2971S 289 Rabbit Cell Signalling 1/1000 (Ser2448) p27Kip1 2552 27 Rabbit Cell Signalling 1/1000 p53 1/500 – sc-6243) 53 Rabbit Santa Cruz (FL-393) 1/5000 p70S6K 2708S 70,85 Rabbit Cell Signalling 1/1000 P-p70S6K 9205S 70, 85 Rabbit Cell Signalling 1/1000 (Thr389) PARP 9542 89, 116 Rabbit Cell Signalling 1/1000 P-Raptor 2083S 150 Rabbit Cell Signalling 1/1000 (Ser792) Rb 9313S 110 Rabbit Cell Signalling 1/1000 p-Rb 8516S 110 Rabbit Cell Signalling 1/1000 (Ser807/811) SQSTM1/p62 8025S 62 Rabbit Cell Signalling 1/1000 (D5E2)
Table 2.1: List of antibodies used
2.2 Cell lines The human ovarian cancer cell lines A2780 (p53 wild type [12]), IGROV-1 (p53 wild type [13]), OVCAR-3 (p53 mutant [12]), and SKOV-3 (p53 null [12]) obtained from American Type Culture Collection (ATCC, Manassas, VA, USA. The above cell lines were maintained in RPMI 1640 medium with 2 mM l- glutamine, 2 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate (Invitrogen, Sydney, Australia) supplemented with 10% heat inactivated fetal bovine serum (FBS) and penicillin–streptomycin (50 U/ml) at 37ºC in a humidified atmosphere containing 5% CO2. Together with human ovarian surface epithelial (HOSEpiC) in OEpiCM (SienCell, CA, USA). The culture media used were all supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mixture (Invitrogen, CA, USA). Primary human umbilical vein endothelial cells, primary human umbilical vein endothelial cells (HUVEC),
Page 71
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Chinese hamster ovarian (CHO) and human embryonic kidney (HEK) cells, used in this study were originally obtained from the American Type Culture Collection (ATCC) and maintained according to their instructions.
2.3 General methods
2.3.1 Dissociation of cells from the culture flask
The following general procedure was used to rapidly remove adherent cell lines from the substratum while maintaining cellular integrity. Cell lines were cultured in T75 cell culture flasks with 15 mL complete medium (DMEM supplemented with 10% FCS, 100 u/mL penicillin G and 100 μg/mL streptomycin sulfate). When cells formed a confluent layer, the complete medium was aspirated and discarded. The cells were washed with 2 mL Dulbecco’s phosphate buffered saline (PBS) by gently rocking the flask. Cells were released from the culture flask by the addition of 2 mL 1X Trypsin-EDTA (ethylene diamine tetra-acetic acid) and incubated at 37°C until the cells were completely dissociated from the substratum. Trypsin action was terminated by the addition 2 mL complete medium. The resulting cell suspension was collected and centrifuged at 1000 rpm (GS-6 centrifuge, Beckman) at room temperature for 5 min.
The supernatant was discarded and the cell pellet resuspended in fresh complete Medium.
2.3.2 Cell counting
The cell number was determined by Trypan Blue exclusion. Aliquot of 0.5 mL cell suspension with 0.1 mL of 0.4% Trypan Blue stain was incubated for 5 min at room temperature. A glass cover slip was placed on a haemocytometer and
Page 72
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
10 µL of the cell suspension added to each well. The cells were then counted in the four outer quadrants and averaged over the two wells.
2.3.3 Freezing adherent cells
Cell lines designated for long-term liquid nitrogen storage were gently detached with 1X trypsin-EDTA as described in section 2.3.2. The cells were resuspended in 2 mL of complete medium to a concentration of 2 - 5 × 106 cells/mL. Aliquot of 500 µL was placed into a cryogenic storage vial and an equal volume of cell freezing medium added. The vial was then frozen slowly at 1°C /min by placing the vial in a NalgeneP TMP Cryo 1 PoPC Freezing Chamber in a -80°C freezer overnight. The following morning, the vial was transferred to liquid nitrogen storage.
2.3.4 Thawing of cryopreserved cells
Cryopreserved cells were thawed quickly in a 37°C water bath. Aliquot of 1 mL was diluted in 10 mL of complete medium and mixed gently. The cells were then centrifuged at 1000 rpm for 5 min, and the supernatant discarded. The cells were then resuspended in fresh complete medium and placed in a T75 cell culture flask.
2.3.5 BCA protein assay
The BCA protein assay is a colorimetric method for detection and quantification of total protein in samples. It is a detergent-compatible formulation based on bicinchoninic acid (BCA). A series of dilutions of known concentration were prepared to make a standard curve using albumin standard (2000 μg/mL) and assayed with the unknowns before the concentration of each unknown was determined based on the standard curve.
Page 73
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
The unknown protein samples were diluted 1:10-1:50 with water. Diluted unknown and standard sample aliquots of 50 μL were placed in triplicate into the wells of a transparent 96-well plate. To each well, 200 μL of the BCA reagent (50:1 ratio of BCA protein assay A: BCA protein assay B) was added. The 96-well plate was sealed with parafilm and incubated at 37ºC / 30 min. The absorbance was read at ODR: 562 using the spectra MAX 250 spectrophotometer and the accompanying SOFT max PTMP software. The standard curve was used to determine the protein concentration of each unknown sample in μg/mL.
2.4 In-vitro Methods
2.4.1 Drug preparation
Monepantel (MPL): The powder was stored at 4ºC. MPL initially dissolved in ethanol and diluted with cell culture media with the final ethanol concentration of 1% (v/v). MPL solution was stored at 4ºC for 30 days. z-VAD-fmk: It is soluble in DMSO (20 mg/mL). Stock solutions kept at −20 °C for 6-8 months.
3-methyladenine (3-MA): It was stored at room temperature and initially dissolved in ethanol and diluted with cell culture media with the final ethanol concentration of 1% (v/v). For more accuracy, it is better to prepare fresh 3-MA solution.
Chloroquine diphosphate salt (CQ): was stored at room temperature and dissolved in water. Solutions of pH 4-6 are stable when heated, but are sensitive to light.
Wortmannin: It was stored at 2-8ºC and soluble in DMSO. Stock solutions kept at −20 °C for 6-8 months. Page 74
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Rapamycin: The powder was stored at −20 °C and soluble in ethanol. 2 mM stock solution in ethanol was stored at -80 °C.
ROD-001: The powder stored at −20 °C for 2 years and is soluble in DMSO. Stock solution in DMSO can be kept at −20 °C for 3 months.
2.4.2 Morphology
We used Giemsa staining (Sigma- Aldrich) to look at the morphology of OVCAR-3, A2780 and HOSE cell lines. Indicated cell lines were treated with desired amount of MPL (0, 5, 10 and 25 µM) for 24, 48 and 72 h. Briefly; cells (104 cells / well) were seeded in 6 wells plate and allowed to adhere overnight on cover slips followed by treatment with MPL for indicated time points. Cells were then fixed with methanol for 10 min. followed by staining with Giemsa (10% in PBS) for 15 min. and washed with tap water. The images were captured with Zeiss, AxioCam, AxioSkop microscope, West Germany. Magnifications were 100x oil immersion lenses or 40x.
2.4.3 Cell viability
The effect of MPL on cell viability of cancer cell lines (A2780, IGROV-1, OVCAR-3 and SKOV-3) and normal cells (HOSE, HUVEC, CHO and HEK) of different origin was measured using the Trypan blue assay. The percentage of viable cells was assessed prior to each experiment (0 – 25 μmol/L MPL for 72 h) using 0.4% (w/v) trypan blue dye exclusion. After trypsinization, cells were mixed with an equal volume of trypan blue solution and counted by hemocytometer. The percentage of viable cells was determined as follows:
% viability = number of viable cells (cell which did not stain) / total number of cells
Page 75
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
For all in vitro and in vivo experiments, cells with >90% viability were used.
2.4.4 Cell proliferation assay (Sulforhodamine B, SRB)
The effect of MPL on in vitro cell proliferation on malignant cell lines (A2780, IGROV-1, OVCAR-3 and SKOV-3) versus normal cell lines (HOSE, HUVEC, CHO and HEK) was assessed using the sulforhodamine B (SRB) assay (Vichai & Kirtikara, 2006). Briefly, cells were seeded in 96-well plates (2000–3000 cells / well) and treated with increasing concentrations of MPL (0 -100 μmol/L) for 72 h. Cells were fixed with 10% ice-cold trichloroacetic acid (TCA) for 30 minutes on ice followed by five washes with tap water. Cells were stained for 15 minutes with 0.4% (w/v) SRB, then dissolved in 1% acetic acid, and washed five times with 1% acetic. Bound SRB was solubilized with 100 μL of 10 mM Tris base (pH 10.5) and the absorbance read at 570 nm. Cell proliferation was calculated using the formula:
% cell proliferation = OD of treated cells / OD of control × 100
Pooled data from at least two experiments are presented as % control (mean ± SEM) where vehicle treated cells is taken to represent 100% proliferation. The IC50 value represented the concentration of the drug that is required to inhibit the proliferation of 50% of the cells compared with non-treated cells.
2.4.5 Cell proliferation assay (MTT)
The rate of replication of MPL treated cancer cell lines was determined by proliferative assay using MTT dye [3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide. The assay was carried out as follows. Briefly, cells seeded in 96-well plates (2000–3000 cells / well) and treated with increasing concentrations of MPL (0 -100 μmol/L) for 72 h.
Page 76
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
MTT dye preparation: dissolve 5 mg/ml of MTT reagent in phosphate buffered saline (PBS) (pH 7.2) then sterilise with filtration.
The cell culture medium was then replaced with 20 µl / well MTT and incubates for 3 h, at 37ºC in a CO2 incubator. After 3 h, the supernatant was removed and 100 µl of dimethylsulfoxide (DMSO, Sigma) was added to all the wells and shake for 20 min to dissolve the formazan crystals. The optical density (OD) was measured at a test wavelength of 562 nm.
2.4.6 Colonogenic assay (colony formation)
In order to determine the effect of MPL on cell integrity following exposure and then withdrawal of the drug from the media, colony formation assay was performed. Briefly, 5×106 cells were plated in 100 mm Petri dishes and allowed to attach overnight. Media were aspirated off and exponentially growing cells were incubated with various concentrations of MPL for 72 h. At this point, the medium was aspirated, the dishes were washed with PBS, and drug free medium was added to each plate. Plates were incubated for two weeks under standard cell culture conditions in an incubator at 37˚C. Following this, plates were gently washed with PBS and cells were fixed with 100% ethanol and stained with a 0.1% solution of filtered crystal violet (w/v). Colonies consisting of more than 50 cells were counted manually. Results are presented as mean ± SEM relative to vehicle treated controls.
2.4.7 Cell cycle analysis
The effect of MPL on the cell cycle was determined using flow cytometry analysis. Briefly, 5 × 105 cells were seeded in 25 cm3 flasks and allowed to adhere overnight, then treated with MPL for 48 h. Cells were collected with trypsinization and pooled with the cells floating in the medium. The cell suspensions were centrifuged, washed with PBS and fixed with methanol.
Page 77
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Washed-cells were resuspended in propodium iodide and ribonuclease-A (diluted in PBS) for 30 min at room temperature (22ºC) and analysed by flow cytometry (Becton Dickinson FACSCalibur).
2.4.8 Western blot analysis
Preparation of buffers/solutions:
(a) Running buffer: 25 mM Tris, 192 mM glycine, and 0.1% SDS; (b) Transfer buffer: 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol; (c) PBS: 0.85% (w/v) NaCI, 0.115% (w/v) of Na2HP04, and 0.02% (w/v) NaH2P04 H20; (d) TBST buffer: 0.2% (v/v) Tween-20 in PBS; (e) and Blocking solution: 5% non-fat dry milk or 5% bovin albumin in TBST.
Preparation of SDS-PAGE gels: SDS-PAGE gels were prepared, using the following formula:
(a) Resolving gel: 375mM Tris-CI (pH 8.8), 6-12 % (w/v) 29:1 acrylamide/bis-acrylamide (Cat. # 161-0156, BioRad), 0.1% (w/v) of SDS, 0.1% (w/v) ammonium persulfate (APS), and 0.1% (v/v) tetramethylethylenediamine (TEMED); and
(b) Stacking gel: 0.125 mM Tris-CI (pH 6.8), 0.1% SDS, 0.05% APS, and 0.33% TEMED. In most cases, 15-well mini gels were pre-casted one day before use, and stored at 4°C.
Preparation of cell lysates:
Cells (1-3 × 106) were plated in 75ml flasks (medium size) overnight in the growth medium and grown overnight at 37°C and 5% CO2. The cells were then treated with MPL (0, 5, 10, 25 µmol/L) for 24 - 72 h. The used medium was Page 78
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
removed by aspiration, and the cells were briefly washed with cold PBS, scrapped with RIPA buffer containing 10% phosphatase inhibitor and protease inhibitor cocktail (Sigma, St. Louis, MO). Lysates were cleared by centrifugation at 13,000g (13,000 rpm) for 30 min and protein concentrations were determined using BCA protein assay (explained at section 2.3.6).
Equivalent amounts of whole cell extracts (equal to 50 µg protein) were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore Corporation, MA, USA).
Blocking and immunoblotting:
After the transfer, the PVDF membranes were incubated in the blocking buffer for 1 h with mild rocking at room temperature. The membranes were washed with mild rocking in TBST 3 times (for 30 min), and then incubated with individual primary antibodies in a Low-Profile Roller (Stovall Life Science Inc., Greensboro, NC) at 4°C overnight. The membranes were washed with TBST 5- 6 times (for 30-45 min), and incubated with appropriate secondary antibodies conjugated to horseradish peroxidase diluted in the blocking buffer at 4°C overnight or 1h at room temperature. The membranes were washed with TBST 5-6 times.
Immunoreactive bands were visualized by incubating individual membrane in the chemiluminescence solution followed by detection (Perkin Elmer Cetus, Foster City, CA, USA). To demonstrate equal protein loading, blots were stripped and re-probed with a specific antibody recognizing GAPDH.
2.4.9 3H-thymidine incorporation assay
3H-thymidine assay was used to determine the effect of MPL on cellular thymidine incorporation (DNA synthesis) (de Fries & Mitsuhashi, 1995). Briefly, cells (104) were seeded onto 24-well plates and treated with increasing
Page 79
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
concentrations of MPL (0-100 μmol/L) for 72 h. For the last 4 h of the incubation, 1 μCi of 3H-thymidine (60 Ci / mM; ICN Biochem) was added to each well. The amount of radioactivity incorporated into cells was determined using a β-scintillation counter. Scintillation fluid was added and counting was performed on a Beckman LS 6000 scintillation counter. Results (mean ± SEM) are expressed as counts per minute (CPM).
2.4.10 Caspases activity assay
Caspase-3 and -8 colorimetric assay kits were used according to the manufacturer’s instructions (Bio scientific, R&D Systems, Gymea, Australia). Briefly, after treatment the cells with indicated concentration of MPL (0, 10 and 25 µM) for 48 and 72 h, cells were harvested, centrifuged at 250 g for 10 min. The cell pellet lysed by the addition of the lyses buffer, then incubated on ice for 10 min followed by centrifugation at 10,000g (10,000 rpm) for 5 min. The supernatant was used to start the enzymatic reaction in 96 well plates based on manufacturer protocol. Supernatant was transferred to a separate microfuge tube. Protein concentration in the supernatant was quantified using a Bio-Rad protein assay kit. The supernatants containing 100 μg of protein were transferred to a 96-well microplate, and lysis buffer was added to each well to make a total volume of 50 μL. Fifty microliters of 2× reaction buffer containing 1,4- DTT at a final concentration of 10 mM and caspase substrates (DEVD-pNA for caspase-3 and IETD- pNA for caspase-8) were added to each well of the microplate. The microplate was incubated at 37°C for 4 hours, and the cleavage of the chromophore pNA from the caspase substrates was measured by reading the absorbance in each well at 405 nm in a microtiter plate reader. Negative controls were generated by omitting the substrates. Each sample was tested in duplicate, and the assays were repeated three times.
Page 80
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
2.4.11 Annexin V / 7-AAD staining
To determine the type of cell death induced by MPL, at indicated time points (24, 48 and 72 h) OVCAR-3 and A2780 cells treated with MPL (0, 10 and 25 µM), washed twice in phosphate-buffered saline and then stained with Annexin V-FITC and 7-AAD (7-Amino-Actinomycin D), according to the manufacturer’s instructions (BD Biosciences, Sydney, Australia). Briefly, after the incubation period, the cells were washed in cold phosphate-buffered saline (PBS). Cells were resuspended in annexin-binding buffer. Cells were diluted in annexin- binding buffer to ~1 × 106 cells/mL. To each 100 μL of cell-suspension, 5–25 μL of the annexin V stain and 7-AAD was added. Stained cells Incubated at room temperature for 15 minutes then washed with annexin-binding buffer. Then, samples were analysed by fluorescence-activated cell scanner (FACScan) flow cytometer (BD FACSCanto II). Using FL1 (annexin V-fluorescein isothiocyanate) and FL3 [7-aminoactinomycin D (7-AAD)] detectors. Annexin V- positive cells were defined as apoptotic, and annexin V-7-AAD-double positive cells were defined as necrotic. Data represent mean ± SEM from three independent experiments combined.
2.4.12 Analysis of inter-nucleosomal DNA fragmentation (DNA Ladder)
Genomic DNA was isolated from cells using apoptotic DNA ladder kit (Millipore), subjected to electrophoresis through 1.5% agarose gel in Tris– borate EDTA buffer, and stained with SYBR® Safe DNA gel stain (Invitrogen, Life technology, Sydney, Australia). Briefly, 2×106 cells were seeded in 75 ml, medium size flasks and allowed to adhere overnight. Following treatment with MPL for 48 h, cells were trypsinized and counted. The pellet of 5-10 × 105 cells was processed according to the manufacturer’s protocol. Briefly, the pellet was gently resuspended in 55 μL of solution #1 (kit component). It was then added with 20 μL of solution #2 (kit component) and was incubated for 60 min. Afterwards 25 μL of solution #3 (kit component) was added, gently mixed and incubated at 50 °C for 3 h. The DNA was precipitated with the kit reagents
Page 81
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
provided and dissolved in 50 μL of resuspension buffer. For detecting the DNA ladder, the extracted DNA samples were run on a 1.5% agarose gel in Tris– acetic acid–EDTA buffer. After electrophoresis, the gel was stained with ethidium bromide (Gibco BRL Co. Ltd., Paisley, Scotland), visualized with a UV light transilluminator, and photographed.
2.4.13 Quantification of acidic vesicular organelles (AVO) by acridine orange (AO) staining
AO is a fluorescent molecule used either to identify apoptotic cell death or autophagy. It can interact with DNA emitting green fluorescence or accumulate in acidic organelles in which it becomes protonated forming aggregates that emit bright red fluorescence (Paglin et al., 2001). Briefly, cells were treated with indicated concentrations of MPL (0, 5, 10 and 25 µM) for indicated time points followed by staining with 1 μM acridine orange for 15 min. Cells were then washed, re-suspended in PBS and subjected to FACS analysis. The green (510–530 nm, FL-1) and red (650 nm, FL-3) fluorescence of AO with blue (488 nm) excitation, was determined over 10,000 events and measured on a FACScan cytofluorimeter using the Cell Quest software (Becton Dickinson, San Jose`, CA, USA). Presented results are as the mean ± SEM of duplicate samples from two experiments (P < 0.05).
2.4.14 Immuno-fluorescence, confocal scanning microscopy
Cells were seeded onto glass cover slips contained in six-well plates in
RPMI medium containing 10% FCS in a humidified, 5% CO2 incubator at 37°C for 24 h. After incubation with indicated concentration of MPL (0, 10 and 25 µM), cells were fixed in 4% paraformaldehyde prepared in phosphate-buffered saline (PBS), Permeabilized with ice-cold 100% methanol, and immuno-stained with indicated antibodies and related secondary. Fluorescence images were
Page 82
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
obtained using OlympusIX71 laser scanning microscopy with 60x oil immersion lenses and Zeiss, Vert.A1, AxioCam, MRm with 40x lenses.
2.4.15 Enzyme-Linked Immunosorbent Assay (ELISA) for p70S6K and phosphorylated form
To quantify the endogenous levels of p70S6K and its phosphorylated form, Path Scan® intracellular p70S6 Kinase (#7038) and P-p70S6 Kinase (Thr389) (#7063) sandwich ELISA kits was used according to the manufacturer’s instructions (Cell Signaling Technology, Sydney, Australia). Briefly, following treatment with 10 µM MPL, cells were harvested, washed with ice-cold PBS, lysed, and then incubated on ice for 5 min. Lysed cells were scrapped and sonicated on ice followed by 10 min microcentrifuge at 4ºC. The lysate was added to wells in quadruplicate and incubated for 2 h at room temperature. Following washing with “wash buffer 1X”, the detection antibody cocktail was added to each well and incubated for 1 h at room temperature. The HRP-linked streptavidin was then added to each well for another 30 min at room temperature. Following the last washes, TMB substrate was added to each well for 10 min and the process terminated by adding stop solution. Absorbance was read at 450 nm. Data represent mean ± SEM from three independent experiments combined (P<0.05).
2.5 In-vivo method
2.5.1 Drug formulation
To prepare the suspension formulation, MPL was suspended in sterile PBS containing 0.5% (w/v) hydroxypropyl methylcellulose (HPMC) and tween 80, yielding to desire final concentrations (25 and 50 mg/ml). The suspension was then stirred for 24 hours at room temperature followed by 20 min sonication for 5 times. MPL suspension was vigorously shaken before the administration to
Page 83
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
the mice. For efficacy experiment, MPL suspension was freshly prepared every second day.
2.5.2 Mice
6 – 8 weeks female BALB/c Nu/Nu nude mice obtained from The Biological Resources and Imaging Laboratory (UNSW, Sydney, Australia) and housed in a pathogen free environment for one week before the commencement of experiments, were fed autoclaved pellets and sterile water ad libitum. Health status of each animal was monitored daily and all experiments were conducted according to protocols approved by the Animal Experimentation Ethics Committee of the University of New South Wales. At the end of the experiment, mice were euthanized by an overdose of Lethabarb.
2.5.3 Cell line
The human ovarian cancer cell line OVCAR-3 used in this study was originally obtained from the American Type Culture Collection (ATCC). Cells kept in RPMI 1640 medium with 2 mM L-glutamine, 2 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate (Invitrogen, Sydney,
Australia) supplemented with 10% heat inactivated fetal bovine serum (FBS) and penicillin–streptomycin (50 U/mL) at 37°C in a humidified atmosphere containing 5% CO2. These cells were processed over time to efficiently grow (100% growth) in the peritoneal cavity of BALB/c nude mice and produce malignant ascites around 3 weeks after inoculation of 10 million cells. The cells isolated from malignant ascites of carrier mice were used throughout the study.
2.5.4 Tumor induction
Sub-cutaneous tumor (SC); following a week of acclimatization, 2.5×106 log- phase growing OVCAR-3 cells suspended in 0.1 ml of matrigel were injected
Page 84
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
subcutaneously into the flank of the animals. When the tumors had grown to approximately 100 mm3, mice were randomised into 3 groups. 5 mice in each group.15 animals totally.
2.5.5 Tumor treatment
Intra-peritoneal injection (IP); mice were treated for 15 days with1 mL/20 g body weight sterile MPL suspension. MPL suspension was injected into the peritoneal cavity of each mouse. The control group received drug free vehicle.
2.5.6 Evaluation of MPL formulation on tumor growth
For SC tumors, body weight and tumor volume were measured 3 times every week and tumor volume calculated using the following formula:
Tumor volume = (shortest diameter) 2 × longest diameter × 0.5
Animals were euthanized when the tumour size reached 1000 mm3. During the course of this investigation, no mice died or became moribund necessitating euthanasia.
2.5.7 Sample collection and analysis
At the end of the treatment period (day 22) and prior to euthanasia, cardiac blood samples were collected. Tumors were excised, weighed and snap frozen in liquid nitrogen for western blot analysis. Tumor weights were presented as mean ± standard error of the mean (SEM).
Page 85
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
2.5.8 Immunohistochemistry
For this analysis, we applied the following process:
A) Deparaffinisation & Re-hydration
1. Section 5µm cut and mounted on SuprPlus or silane coated slides 2. incubate slides on an oven, 58° C for 15 -20 mins. 3. Dewax sections in 2 changes of xylene, 10 minutes each 4. Tap off excess liquid and place slides in absolute ethanol for 5 mins. 5. Tap off excess liquid and place slides in 95% ethanol for 5 mins. 6. Tap off excess liquid and place slides in 75% ethanol for 5 mins. 7. Wash slides in distilled water
B) Antigen retrieval
Microwave Method
1. Immerse slides into plastic staining dish containing antigen retrieval solution (citrate buffer or sodium citrate buffer) and Place the lid loosely on the staining dish, Then place staining dish with slides into other plastic tray with water, Check the level of water in the tray and only need to ¾ cover the staining dish. 2. Adjust the power level of the microwave; microwave on med/high for 2-5 mins ( wait until the solution comes to the boil), and then on low for 4-6 mins, (depend on power of microwave). 3. Place the staining dish at room temperature and allow slides to cool at room temperature for 20 min. 4. Wash slides in water 5. place slide in tris buffer
Page 86
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Water-Bath Method
1. Pre-heat water bath with staining dish containing Sodium Citrate Buffer or Citrate Buffer until temperature reaches 90-95 C. 2. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes. 3. Remove the staining dish to room temperature and allow the slides to cool for 20 minutes. 4. Rinse sections in tris buffer 5. Block sections with for 30 minutes.
C) Staining procedure
1. Block peroxidase blocking solution Apply 3% hydrogen peroxide/ in Methonal to cover specimen. Incubate for 15 mins. Rinse gently with distilled water. 2. Place slides in TBS bath for 5 mins. 3. Block specimen with 10% milk/TBS for 15mins. 4. Rinse gently with distilled water and place slides in TBS for 5 mins. 5. Draw a circular around the tissue sections with a Pap Pen, 6. Dilute Primary antibody (concentration depend on each antibody) in 1% BSA/TBS solution 7. Place slide in a incubation-Box and place 50-100 diluted primary antibody to each slid, Incubate sections with primary antibody for 1 hour at room temperature or overnight at 4 C. 8. Rinse with TBS and place in TTBS for 5 min x 2. 9. Incubate multi-LINK with specimen for 15 mins. 10. Rinse with TTBS and place in TBS for 5 mins x 2 11. Incubate Strepavidin-Peroxidase with specimen for 15 mins. 12. Dalute DAKO-DAB wih buffer, (20µ DAB in 1ml buffer) and keep in dark. 13. Rinse with TBS and place in TTBS for 5 mins x 2
Page 87
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
14. Incubate specimen with diluted DAKO liquid DAB for 2-8 mins (check colour). 15. Place slides in water.
D) Counterstain
16. Haemotoxylin stain for 2 mins.
17. Wash in a wash bath filled with tap water for 10 mimns. 18. Tap off excess liquid and 2 -3 dips in 1% Acid/alcohol solution. 19. Rinse in water 20. immerse in SChtt’s blue solution for 2-5 min (depend on type of tissue) 21. Place in water for 1 mins. 22. dehydrate in 70%, 95% and 100% for 3 mins each 23. Place in xylene 24. Mount the slides with DPX mounting solution in chemical Hood and dry up at room temperature o/n.
2.5.9 Statistical analysis
GraphPad Prism 6 version (GraphPad Software, Inc., La Jolla, CA) was used to create the x-y scatter plots or bar graphs with mean ± standard error (S.E.). When needed, the non-linear regression curves (best-fit curves) were generated over x-y scatter plots. The statistical analyses were performed using the one-way ANOVA (analysis of variance) with the Dunnett post hoc tests. In vitro quantitative variables were compared using the Student’s t-test. P value less than 0.05 was considered to be statistically significant.
Page 88
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Chapter 3: Anticancer properties of aminoacetonitrile derivative, monepantel (ADD1566) in ovarian cancer
First publication: Farnaz Bahrami, David L. Morris, Lucien Rufener, Mohammad H. Pourgholami, Anticancer properties of novel aminoacetonitrile derivative monepantel (ADD 1566) in pre-clinical models of human ovarian cancer, American Journal of Cancer Research, 2014; 4(5): 545–557
3.1 Aim
The realization that MPL is a very well tolerated agent in mammals coupled with the fact that the other major antiparasitic treatments such as the macrocyclic lactones, imidazothiazoles and in particular the benzimidazole carbamates have all shown some degree of anticancer activity encouraged us to evaluate MPL in laboratory based anticancer tests. Our work on benzimidazole carbamates (BZD) has shown potent anticancer properties for this group of agents and in particular for albendazole in ovarian cancer (Pourgholami et al., 2010; Pourgholami et al., 2006). Similarly, macrocyclic lactones and imidazothiazoles have also been shown to exert anticancer activity in pre-clinical models of cancer (Naito et al., 1998; Towle et al., 2001). We therefore placed MPL in a systematic progressive series of tests starting from visualization of its microscopic effects on cells, to cellular and molecular. Results obtained conclusively demonstrate that MPL has anticancer properties. The effects are characterised by inhibition of cancer cell proliferation and colony formation, induction of cell cycle arrest, interference with the expression of cyclins and cyclin dependent kinases (CDKs), inhibition of DNA synthesis and cleavage of PARP-1. This is the first report to describe the anticancer properties of monepantel. Page 89
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
3.2 Results
3.2.1 MPL decreases cell proliferation of ovarian cancer cells
The ovarian cancer cell lines (OVCAR-3 and A2780) were selected based on their different molecular characteristics and clinical behaviour. In these cells, addition of MPL to media bathing the cells led to concentration-dependent reduction of cell viability. As presented in Figure 3.1, MPL treatment profoundly impaired the survival capacity of cancer cells.
Figure 3.1: MPL inhibits growth of epithelial ovarian cancer cells Human ovarian cancer cell lines OVCAR-3 and A2780 were grown in 6 well tissue culture plates under standard cell culture conditions in the presence of MPL (0, 5, 10, 25 µM) for 72 h. Cells were then stained with Giemsa, washed and photographed (40x magnification; using Leica DM IRB light microscope). Experiment repeated twice with the same result, n=3.
Page 90
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
3.2.2 MPL decreases cell viability of ovarian cancer cells
Trypan blue exclusion assay was used to quantitatively determine the effect of MPL on cell viability. Ovarian cancer cell lines (OVCAR-3, A2780, SKOV-3 and IGROV-1) together with normal (non-malignant) human cells (HOSE, CHO, HEK and HUVEC) were all examined for their response to MPL (Figure 3.2). As demonstrated, clear difference between the effects of MPL on cancer versus normal non-malignant cells was found. The treatment of cancer cells with MPL resulted in concentration-dependent reduction of cell viability, whereas the viability of normal cells was either hardly affected (HOSE) or far less (CHO) affected by MPL.
Page 91
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 3.2: Effect of MPL on cell viability is cell specific Effect of MPL on cell viability was determined using trypan blue dye exclusion assay. Treatment of ovarian cancer cells (top panel) with MPL (0, 5, 10, 25 µM) for 72 h, reduced cell viability and induced cell-death in a concentration- dependent manner. Normal (non-malignant) cells exposed to the same concentrations of MPL over the same period were far less susceptible (bottom panel). The effect of MPL on the viability of human ovarian surface epithelial (HOSE) cells was minimal (p <0.001 at all concentrations compared to OVCAR- 3 cells). Data represent mean ± SEM (n=2).
Page 92
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
3.2.3 MPL decreases cell proliferation (based on cellular protein content) of ovarian cancer cells
Based on the above results, we next examined the effect of MPL on cell proliferation using the SRB assay and based on live cells protein. Treatment of the above mentioned cells with MPL suppressed proliferation of all 4 ovarian cancer cell-lines used in this study (Figure 3.3). The inhibitory effect of MPL on proliferation was concentration-dependent with IC50 values ranging from 4.4 ± 0.27 μM to 31.18 ± 0.76 μM. Comparison of the IC50 values demonstrates that HOSE cells are almost 10 fold less sensitive to MPL than human epithelial ovarian cancer cells such as OVCAR-3 (74.8 ± 7.7 µM for HOSE compared to 7.2 ± 0.2 µM for OVCAR-3). It is evident from these results that the ovarian cancer cell lines are quite sensitive to the antiproliferative effects of MPL whereas, normal cells and in particular the HOSE cells are minimally affected (p < IC50 value for MPL in HOSE compared to OVCAR-3 using student’s t-test). Whereas the IC50 value (10.5 ± 0.4 μM) for A2780 with wild type p53 was quite similar to that for p53 mutant OVCAR-3. The highly chemoresistant p53 null SKOV-3 cells with an IC50 value of 31.2 ± 0.7 μM were found to be the least sensitive of the ovarian cancer cells to MPL which is some 5 to 7 fold higher than values for the other cells.
Page 93
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 3.3: MPL suppresses proliferation (based on cell’s protein measurement) of ovarian cancer cells The impact of MPL (0, 5, 10, 25, 50 and 100 µM) on cell proliferation was assessed using the SRB assay. Control (vehicle treated) cells were taken to present 100% proliferation and values for the MPL treated groups are expressed as percentage of control (mean ± SEM, n≥3).
Page 94
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
3.2.4 MPL decreases cell metabolism of ovarian cancer cells
Based on the above results, we next examined the effect of MPL on cell proliferation through MTT assay and based on live cells’ metabolism. Treatment of the above-mentioned cells with MPL suppressed proliferation of ovarian cancer cell-lines (OVCAR03 and A2780) (Figure 3.4). The inhibitory effect of MPL on proliferation was again concentration-dependent.
Page 95
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 3.4: MPL suppresses metabolism of ovarian cancer cells The impact of MPL (0, 5, 10 and 25 µM) on cell proliferation was assessed using the MTT assay. Control (vehicle treated) cells were taken to present 100% proliferation and values for the MPL treated groups are expressed as percentage of control (mean ± SEM, n=3).
Page 96
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
3.2.5 MPL inhibits colony formation
In order to assess the effect of MPL on the reproductive integrity of cell lines to establish colonies, we next investigated the clonogenic activity of MPL treated cells (OVCAR-3 and A2780). Following 72 h exposure to varying concentrations of MPL (0, 5, 10 and 25 μM), it was found that MPL exposure leads to profound suppression of colony formation by these cells. Higher concentrations of MPL (> 10 µM) led to almost complete loss (p<0.001 versus control) of their clonogenic capacity (Figure 3.5).
Page 97
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 3.5: MPL effects on the colony formation activity of ovarian cancer cells Following incubation of OVCAR-3 and A2780 cells with MPL (0, 5, 10, 25 µmol/L) for 72 h, cells were washed and then transferred to agar plates, cultured with RPMI growth medium (drug free) and incubated under standard conditions for 14 days. Cells were then fixed with 100% methanol and stained with 0.1% crystal violet. Each experiment was repeated twice, n=3. Colonies (Clusters of cells greater than 50) were counted manually. Number of colonies counted for different experimental groups (MPL treated) is expressed as percentage of the control.* = p < 0.05; ** = p < 0.01 and ***= p < 0.001 as compared to control (vehicle treated) group using Student t-test.
Page 98
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
3.2.6 Antiproliferative activity of MPL is independent of acetylcholine signaling
The nematicidal effect of MPL has been linked to its interaction with the nicotinic subtype MPTL-1 nematode receptor. To determine if the MPL antiproliferative effects are also mediated via acetylcholine receptors, cells were pre-treated with a range of cholinergic / anticholinergic agents. On this basis, cells were initially treated with nicotine and the stable long acting synthetic nicotinic agonist carbachol. Compared to control vehicle treated cells, these nicotinic receptor agonists did not exert an effect of their own nor did they alter the extent of MPL antiproliferative activity (Figure 3.6). Activity at nematode nicotinic acetylcholine receptors (nAChRs) has been considered to account for a major part of the mechanism of action of several classical anthelmintic agents (Ruiz-Lancheros, Viau, Walter, Francis, & Geary, 2011). To extend these findings, in the next series of experiments, cells were pre-treated with cholinergic receptor antagonists ranging from the broad cholinergic antagonist atropine and then followed by the more nicotinic selective mecamylamine, tubocurarine and progressed to finally using the highly selective irreversible nAChR7α antagonist α-bungarotoxin (Rao, Correa, & Lloyd, 1997). The antagonist concentrations used were selected from literature where a positive receptor mediated effect had been reported. It is evident from our results that unlike the nematicidal effects, the antiproliferative activity of MPL in cancer cells is not mediated through the nicotinic signalling pathway.
Page 99
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 3.6: MPL-antiproliferative effect is not mediated through acetylcholine nicotinic receptor Pre-treatment (30 min) of OVCAR-3 cells with nicotinic agonist (nicotine, carbachol) or nicotinic antagonists (atropine, mecamylamine, tubocurarine or α- Bungarotoxin) at the indicated concentrations did not change the antiproliferative effects of MPL under the cell culture conditions. Proliferation was assessed using SRB assay. Data represent mean ± SEM, n=2.
Page 100
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
3.2.7 MPL induces G1 cell cycle arrest
To find out the mechanism/s through which MPL inhibits growth of cancer cells, using flow cytometry (FACS) we next examined the effects of MPL on cell cycle progression, using the same concentrations as above (0, 5, 10 and 25 µM). It was found that MPL interferes with the cell cycle progression resulting in higher number of cells in the G1 phase in time and concentration-dependent manner (Figure 3.7). Accumulation of cells in the G1 phase was accompanied by sharp decline of percentage of cells in the S phase. In OVCAR-3 cells the percentage of cells in the G1 phase significantly (p< 0.05) increased from 65.8 ± 6.5 (control) to 87.7 ± 2.2 (MPL 25 µM). Consistent with this, the percentage of cells in the S phase declined from 8.3 ± 1.9 in vehicle treated cells to 2.5 ± 0.5 in 25 µM MPL treated cells (p<0.05). The effects of various MPL concentrations on the cell cycle and percentage of cells in each phase are also summarized in Figure 3.7.
Page 101
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
(A)
OVCAR-3
SIOY-3.006 Controi i OY~ .OOS ~ ,.._C> Control C> ... 0 M 3 I .. M3 1 ~ .. ~ ~~ M4 ~ & & <..> <..>~ ~
~ ~ e
1: FL2..._ FL2..._ -:I 0 IOIOV~007 2S I OY-3.008 (.) ~ 10 JJM Q) ~ (.) ~ M 3 I ~ a ~8 & J~ <.> ~ ~
2 ~
<>
Fl2""' Fl2..._ Fluorescence Intensity
OVCAR-3 Cell cycle distribution (% of total)
MPL (IJM) Sub-G1 G1 s G2-M
0 0.51± 0.08 65.85 ± 6.50 8.36 ± 1.89 23.51 ± 7.9
5 0.32 ± 0.08 70.75 ± 6.85 8.67 ± 2.13 20.3 ± 8.6
10 0.23 ± 0 73.87 ±5.44 6.98 ± 0.81 18.94±6.24
25 0.48±0.07 87.76±2.22 2.54±0.55 6.2±2.9
Page 102
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
(B)
A2780
~ II !::! 5J,JM ~ Control M3 1 M 3 I ~ j~
$j!
~
1:: -::I A-1 0 (.) ~
Q) (.) 2
.. ~ v~ $j!
~
~
FL2..A Fluorescence Intensity
Cell cycle distribution (% of total)
MPL (IJM) Sub-G1 G1 s G2-M
0 1.43 ± 0.23 52.02 ± 1.82 10.43 ± 3.4 37.89 ± 3.45
5 1.045 :!:0.02 55:!: 0.01 9.65:!: 0.25 33.98:!: 0.1 8
10 2.13 :t 0.16 58.83 ±1.23 9.46 ± 1.5 28.85 ± 1.6
25 3.07±0.05 63.14±2.7 7.02 :t 1.3 25-41 :t 0.3
Page 103
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 3.7: MPL effects on cell cycle progression of human ovarian cancer cells (A) And (B) OVCAR-3 and A2780 cells treated with either MPL or the vehicle (control group) for 48 h were harvested, washed, digested and stained with propidium iodide and analysed by flow cytometry. Distribution of cells in the various phases of the cell cycle (G1, S and G2/M) are presented as percentage, with values (mean ± SEM, n=2).
Page 104
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
3.2.8 MPL down regulates the expression of cyclins and cyclin - dependent kinases to induce G1 cell cycle arrest
As the treatment of these cells with MPL induces G1 arrest, we next assessed the effect of MPL on cell cycle regulatory molecules notably cyclins D1, A and E2 together with their associated cyclin-dependent kinases CDK2 and CDK4 and the CDK inhibitors p27 Kip1 and p53 (Figure 3.8). MPL-induced G1 cell cycle arrest was accompanied by diminished cell cycle regulator proteins, like cyclin D1, cyclin A and related CDK2 and CDK4 along with changes in p27 Kip1 and p53 expressions (Figure 3.8). These results further confirm the induction of G1 cell cycle arrest after treatment with MPL.
Page 105
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 3.8: MPL modulates expression of the G1 cell cycle regulatory proteins Western blot of lysates prepared from cells treated with MPL (0, 5, 10, 25 µmol/L for 48 h) were analysed for the expression of cyclin D1, CDK4, cyclin E2, cyclin A, CDK2, p27kip1 and p53 proteins. The housekeeping gene (GAPDH) was used to confirm similar protein loading and blot transfer.
Page 106
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
3.2.9 MPL inhibits thymidine incorporation
Impairment of the cell cycle machinery leading to cell cycle arrest at G1 inevitably leads to reduced DNA synthesis, which in turn attenuates cellular thymidine incorporation. Besides this, cyclins, their kinases together with their inducers and inhibitors are all known to regulate thymidine incorporation, which is an essential process for the cell proliferation machinery to operate. To determine the effect of MPL on DNA synthesis in these cells, we measured thymidine incorporation. As depicted in Figure 3.9, MPL treatment conferred profound reduction in 3H-thymidine incorporation as judged by reduced counts per minute (CPM) produced by the scintillation counter. Under the influence of 25 µM MPL, CPM was reduced by 75.6 % in OVCAR-3 and 84.2% in A2780 cells (in both cases p<0.001 when compared to vehicle treated cells). The results confirm the above findings on MPL-induced cell cycle arrest and inhibition of proliferation in ovarian cancer cell lines.
Page 107
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 3.9: MPL inhibits thymidine incorporation Thymidine incorporation in MPL (0, 5, 10, 25, 50 and 100 µmol/L) treated cells. The y-axis presents the actual counts per minute (CPM). Data represent mean ± SEM, n=2.
Page 108
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
3.2.10 MPL induces PARP-1 cleavage
PARP helps cells to maintain their viability and hence cleavage of PARP facilitates cellular disassembly and serves as marker of cell death (Oliver et al., 1998). Therefore, to determine if the MPL-induced loss of viability, proliferation and clonogenic activity is accompanied by cleavage of PARP-1, using western blot analysis we examined lysates of MPL-treated cells for the expression of cleaved PARP-1 after 24, 48 and 72 h of drug treatment. Under cell culture conditions, MPL induced cleavage of PARP in both OVCAR-3 and A2780 cells. This observation is in line with the above presented data showing inhibition of cell proliferation and colony formation activity together with suppression of thymidine uptake by these cells. MPL-induced PARP-1 cleavage was more evident in A2780 than in OVCAR-3 cells. Whereas in the former, cleaved PARP-1 was clearly detectable at 24 h, in OVCAR-3 cells, cleaved PARP-1 was only detected in 25 µM MPL treated cells in 48 and 72 h samples (Figure 3.10). PARP-1 participates in DNA repair, genomic integrity, and cell death (Pagano et al., 2007).
Page 109
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 3.10: MPL induces PARP-1 cleavage Immunoblot analysis for the detection of PARP-1 and cleaved PARP-1 in cells treated for the indicated period of time (24-72 h) with MPL (0, 5, 10, 25 µmol/L). . The housekeeping gene (GAPDH) was used to confirm similar protein loading and blot transfer.
Page 110
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
3.3 Discussion
This study provides for the first time a report of anticancer properties of MPL. Here we found that MPL presented potent anticancer activity in various cancer cell lines, having IC50 values of ≈ 4 to 30 µM for inhibition of cell proliferation and viability of different ovarian malignant cell lines. In contrast, control normal cell lines (HOSE and HUVEK) were quite resistant to MPL exposure, with IC50 > 70 µM and in lesser degree of resistance for CHO and HEK with IC50 more than 30 µM. Notably, both cell lines did not resume proliferation even after 3 weeks post-treatment in a drug-free medium. All these results indicate that this compound is capable of inducing permanent and irreversible suppression in preclinical studies of ovarian cancer cell lines; moreover, our findings suggest that sensitivity of cancer cells to MPL differs from that of normal cells.
Furthermore, in the present study, we observed that the percentage of cells in G1 phase of cell cycle increased which demonstrated the inhibitory effect of MPL is resulted from the cell cycle arrest at G1 phase. In normal condition, cell cycle progress is regulated by certain controllers, named CDKs (Malumbres & Barbacid, 2009). Normal cells need cyclins for their activation. Among cyclines, CDK2 and 4 are essential for the G1 transition stage. While cyclin A and E are responsible for activation of CDK2, cyclin D1 helps to activate CDK4 and CDK6 (Malumbres & Barbacid, 2009). Protein expression profile revealed that MPL obviously suppressed expression of cyclin D1 and cyclin A and related cyclin dependent kinases; CDK4 and CDK2, in a concentration- and time-dependent manner. We observed that MPL regulated p27kip1 expression, as well, and it did not change the expression of cyclin E2 in OVCAR-3; however 25 µM MPL caused up-regulation in A2780. Therefore, the effect of MPL on the cell cycle profile in ovarian cancer cells is to be summarized as follows: Reduced expression of cyclin A and D1 result in decreased activity of CDK2 and CDK4, respectively. This accounts for G1 halt. Negative regulators, such as p27kip1 as CDK inhibitors support this phenomenon, as well (Nakayama & Nakayama, 2006). Based on different studies both cyclin D1 and E2 positively regulate cell Page 111
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
cycle, however for G1 cell cycle arrest, down regulation of cyclin D1 would be enough. In 2002 Bowe and his group showed that cyclin E2 compensates cyclin D1 down regulation (Bowe, Kenney, Adereth, & Maroulakou, 2002). On the other hand, Masamha and Benbrook demonstrated that in ovarian cancer cells irrespective of p53 status, loss of D1 and increased cyclin E2 expression still cause cell cycle arrest at the G1 phase (Masamha & Benbrook, 2009). All these observations suggest that up-regulation of cyclin E1 after treatment with MPL would be consequences of cyclin D1 down-regulation and G1 cell cycle arrest.
This conclusion is supported by our observation about the thymidine uptake, which is resulted from the fact that MPL suppresses DNA synthesis and replication. Since decision the cell proliferation is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle; detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments (Cecchini, Amiri, & Dick, 2012). Our results also demonstrate that MPL inhibites thymidine incorporation and it cleaves PARP-1, which both are consequences of MPL induced inhibition of cell proliferation and cell cycle arrest. It is proved that PARP is essential for live cells to keep their vitality, thus PARP cleavage move the cells toward death (Chaitanya, Steven, & Babu, 2010). Furthermore, our results emphasise the higher sensitivity of A2780 cells to inhibitory effects on MPL in comparison to OVCAR-3 groups. This observation can be related to a different type of p53 in these cell lines.
While the anthelmintic activity of MPL is related to its engagement to α-subunit analogue of nematode nAChR, our data indicates that the anti-tumour effect of MPL is not mediated through AchR, and blocking specific (α7-nAchR) or non- specific acetylcholine receptors cannot prevent anti-proliferative effects of MPL on cancer cells.
Page 112
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Conclusion:
As a summary, our results present the anticancer potential of MPL as an aminoacetonitrile derivative, in ovarian cancer in-vitro models. We have shown that MPL reduces the expression of associated cyclin-dependent kinases, CDK2 and CDK4 activity by suppressing cell cycle regulatory proteins cyclin D1 and A, culminating in G1 cell cycle arrest. Furthermore, MPL by preventing DNA synthesis followed by cleavage of PARP-1 resultes in suppression of ovarian cancer cell lines’ proliferation in-vitro.
Page 113
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Chapter 4: Monepantel induces autophagy in human ovarian cancer cells through disruption of the mTOR/p70S6K signalling pathway
Second Publication: Farnaz Bahrami, David L. Morris, Ahmed Mekkawy, Lucien Rufener, Mohammad H. Pourgholami, Monepantel induces autophagy in human ovarian cancer cells through disruption of the mTOR / p70S6K signalling pathway, American Journal of Cancer Research, 201; 4 (5): 558-71
4.1 Aim
We have recently shown for the first time that MPL exerts anticancer effects in a wide variety of cancer cells (F. Bahrami, Morris, Rufener, & Pourgholami, 2014). In particular, we found that, under cell culture conditions, MPL inhibits growth, proliferation and colony formation of ovarian cancer cell lines in a time and concentration-dependent manner. Further studies revealed that, MPL interferes with the cell cycle progression and arrests cells in the G1 phase of the cell cycle. In line with this, we observed activation of PARP-1 by MPL, suggesting that the MPL-induced cell death may be an apoptotic mediated process. Additionally, we found that the anticancer effects of MPL are not nAChR dependent thus suggesting the involvement of other molecular pathways. These observations prompted the design of the current study where we looked at the mechanisms behind the anticancer activity of MPL. Interestingly, we found that MPL does not induce apoptosis but rather causes persistent autophagy through the deactivation of the mTOR/p70S6K signalling pathway.
Page 114
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
4.2 Results
4.2.1 MPL reduced the expression of caspase-3 and -8
We have previously shown the inhibitory effect of MPL on different human tumour cell lines and observed that ovarian cancer cell lines are sensitive to MPL with IC50 values of 7.2 ± 0.2 µM (OVCAR-3) and 10.5 ± 0.4 μM (A2780). Our previous results showed MPL susceptibility was associated with induction of cleaved PARP-1, a molecule that is vital in DNA repair, genomic integrity, and cell death (apoptosis or necroptosis) (Chaabane et al., 2013). We therefore looked for the effect of MPL-induced active caspase-3 and caspase-8, which are apoptosis indicators. Western blot analysis was used to assess the levels of cleavage of procaspase-3 and procaspase-8 to active forms. MPL treated cell lines failed to show active caspase-3 and -8 (Figure 4.1).
Page 115
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 4.1: MPL reduced the expression of caspase 3 and 8 Western blot of lysates prepared from OVCAR-3 and A2780 cells treated with MPL (0, 5, 10, 25 µM for 48 and 72 h) were analysed for the expression of caspase-3 and caspase-8 proteins. The housekeeping gene (GAPDH) was used to confirm similar protein loading and blot transfer.
Page 116
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
4.2.2 MPL decreased caspase-3 and -8 activation
Quantitative analysis of those caspases activities by colorimetric assay confirmed that MPL treatment has led to significant reduction of both caspases in OVCAR-3 cell line. The percentage of reduction of caspase-3 was 23.1 ± 0.4 (P=0.005) and 31.42 ± 1.68 (P=0.018) after 72 h treatment with 10 and 25 µM MPL, respectively. Similarly, the percentage reduction of caspase-8 after 72 h treatment with 10 µM MPL was 12.9 ± 4.9 and with 25 µM MPL was 41 ± 2.6, P=0.017 (Figure 4.2).
Page 117
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 4.2: MPL decreased caspase-3 and -8 activation
The impact of MPL (0, 10 and 25 µM) on caspase-3 and -8 activities was assessed in MPL-treated OVCAR-3 cells for the indicated time points (48-72 h). Data represent mean ± SEM, n=3. For statistical comparisons, each drug treated group was compared with the control group using Student’s t-test.
Page 118
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
4.2.3 Cell death induced by MPL is not caspase-dependent
For further proof, cells (OVCAR-3 and A2780) were pre-treated with the pan- caspase inhibitor z-VAD-fmk and the effect on cell death induced by MPL (0, 10 and 25 µM) was assessed, using the SRB assay. As seen in figure 4.3, addition of z-VAD-fmk did not block the antiproliferative effect of MPL, indicating that the MPL-induced cell death is not caspase-dependent.
Page 119
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
Figure 4.3: Cell death induced by MPL is not caspase-dependent
Cell viability estimated by SRB assay in OVCAR-3 and A2780 cell lines after treatment with MPL (0, 10, 25 µM) 30 min. after pan-caspase inhibitor zVAD- fmk (5 µM) up to 72 h. Data represent mean ± SEM, n=3.
Page 120
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
4.2.4 MPL-induced cell death is not mediated via apoptosis
To find out the involved programmed cell death induced by MPL, Annexin- V+/7-AAD+ labelling was performed. Interestingly, we observed a concentration- dependent increase in the extent of Annexin-V+/7-AAD+ labelling (Late apoptosis or necrosis) after 48 and 72 h of treatment with 10 and 25 µM MPL. After 72 h treatment with 25 µM MPL the percentage of dead cells (Annexin- V+/7-AAD+) compared with control increased from 0.95 ± 0.05 to 21.25 ± 2.15, P=0.0136 (22 fold increase) in OVCAR-3. 10 µM MPL had no significant changes. In A2780 cells, compared with control 10 µM MPL led to a 4.5 fold increase in the percentage of dead cells (from 3.1 ± 0.3% to 14.2 ± 1.7%, P=0.0068) and with higher concentration of MPL (≥ 25 µM) peaked to 7.6 fold after 72 h treatment (From 3.05 ± 0.55% to 23.4 ± 1.6%, P=0.0046) (Figure 4.4). The percentage increase of Annexin-V+ alone (indicator of early apoptosis) in both cell lines was not significant.
Page 121
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
(A)
OVCAR-3
a::- ...J 0 ...... -CD
ql
.r:. N ......
Q4 •• .... ·-~ Annexin-V (530/30)
OVCAR-3
.24h * B48h B72h
*
0 10 25
Page 122
Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer
(B)
A2780
.c: ...... N
II_SlO'»~
Annexin-V (530/30 )
A2780 + ";- 30 ..CCI .24h ** ll48h ! ~ 20 B72h =+ 41 > u c 0 X 10 41 -~ 0 c c MPL (~M) Page 123 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.4: MPL-induced cell death is not mediated via apoptosis (A) And (B) Cells treated with MPL (0, 10 and 25 µM) for the indicated time points (24, 48 and 72 h), stained with Annexin V / 7-AAD and cell death rates were analysed by FACS. Data represent mean ± SEM, n=3. Page 124 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.5 MPL doesn’t induce DNA fragmentation To evaluate the influence of MPL on cell-concentration, internucleosomal DNA fragmentation assay was performed. Treatment with MPL failed to cause DNA fragmentation (Figure 4.5). Put together, these data suggest that MPL- induced cell death mediated through inhibition of caspases activation and a non-apoptotic process. Page 125 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.5: MPL doesn’t induce DNA fragmentation DNA ladders were detected on 1.5% of agarose gel after OVCAR-3 and A2780 cells were treated with 25 μM MPL for 48 h. A representative from three independent experiments is shown, n=3. Page 126 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.6 MPL induces vacuole formation To determine the anticancer effect of MPL, we next examined other cellular responses associated with cell death under MPL treatment and in particular autophagy. Figure 4.6 shows representative examples of both treated and untreated cell lines. After 72 h treatment with 10 and 25 µM MPL, significant elevation in the number of visible vacuoles in malignant cells was observed, while the normal cells (HOSE) were minimally affected. Compared with the control group, the ultrastructure of giemsa stained OVCAR-3 and A2780 cells treated with MPL up to 72 h showed morphological changes in the whole cytoplasm and membrane, including loss of plasma membrane integrity and obvious vacuole formation. The vacuolization was much less pronounced in MPL-treated normal human ovarian surface epithelial cells (HOSE) with no sign of changes in membrane integrity or cell death. This dramatic vacuolization of the cytoplasm without apparent loss of nuclear material is consistent with the described macrostructure of autophagy (Kroemer et al., 2009). Page 127 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.6: MPL induces vacuole formation Human ovarian cancer cell lines (OVCAR-3 and A2780) and Normal human ovarian surface epithelial (HOSE) were grown in 6-well tissue culture plates under standard cell culture conditions in the presence of MPL (0, 10, 25 µM) for 72 hours. Cells were then stained with giemsa, washed and photographed under Leica DM IRB light microscopy (magnification 100x) with oil immersion lenses. A representative from two independent experiments is shown, n=2. Page 128 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.7 Acidic vacuoles induced by MPL is concentration-dependent Having seen that MPL induces vacuole formation, we next performed FACS analysis of AO stained vacuoles on MPL treated cells. Based on the work of Paglin (Paglin et al., 2001), we used the red to green ratio as an indicator of acidic vacuolar organelle (AVO) accumulation and therefore autophagic progression. Quantification of AVO has showed a concentration-dependent increase of AVO’s in both OVCAR-3 and A2780 cells (Figure 4.7). Treatment with 25 µM MPL up to 72 h, led to 12 fold increase vs. control and the percentage of the bright red fluorescence intensity (y-axis) increased from 0.15 ± 0.05 to 1.8 ± 0.3, P=0.32 in OVCAR-3 cells. In A2780 the percentage of increase was from 0.3 ± 0.1 to 13.55 ± 0.26, P=0.0004 (45 fold increase). These data suggest that treatment with indicated concentration of MPL leads to enlargement of the endosomal / lysosomal system and induction of AVOs in ovarian cancer cells. Page 129 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.7: Acidic vacuoles induced by MPL is concentration-dependent The percentage of bright red fluorescence (FL3)-positive cells was evaluated by AO staining and FACS analysis in OVCAR-3 and A2780 cells untreated or treated with MPL (0, 5, 10, 25 µM) at different time points (48 and 72 h). Results are the mean ± SEM, n=3. Page 130 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.8 MPL induces autophagy To determine the possible autophagic nature of the MPL-induced compartments, we next evaluated the effect of MPL on the intracellular localization of microtubule-associated protein 1 light chain 3 beta (LC3B), a specific marker of autophagosomes (Kadowaki & Karim, 2009). Autophagy is associated with LC3B-I being modified to a membrane-bound form, LC3B-II, which is relocated to the autophagosomal membranes during the process of autophagy (Barth, Glick, & Macleod, 2010; Mizushima & Yoshimori, 2007). As shown in figure 4.8, representative fluorescence micrographs show a punctate pattern of LC3B-II. After 48 h treatment with MPL (0, 10 and 25 µM), a marked elevation in the number of cells with visibly increased punctate fluorescence, particularly in the peri-nuclear region of the cytoplasm was observed. This suggests that MPL induces autophagosome formation in these cancer cell lines. Page 131 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.8: MPL induces autophagy Human ovarian cancer cell lines (OVCAR-3 and A2780) were grown in 6 well tissue culture plates under standard cell culture conditions in the presence of MPL (0, 10, 25 µM) for 48 h. Cells were washed, fixed and incubated with primary antibodies in 1% BSA followed by related secondary antibodies in 1% BSA. Fluorescence images were observed and collected under Olympus IX71 laser scanning microscopy, magnification 60x with oil immersion lenses. The experiment was repeated once with identical results. A representative from two independent experiments is shown, n=2. Page 132 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.9 MPL regulates autophagy related proteins To further verify that MPL does induce autophagy, we assessed the effect of MPL on the conversion of the cytoplasmic form of LC3B-I protein (18 kDa) to the pre-autophagosomal and autophagosomal membrane–bound form of LC3B- II (16 kDa). As shown in figure 4.9, increasing concentrations of MPL induced accumulation of both the unprocessed LC3B-I, and the processed form (LC3B- II). Immunoblot analysis revealed that MPL causes a time and concentration- dependent increase in the levels of LC3B-II proteins. Of note, in A2780 cells after 48 and 72 h treatment with 10 µM MPL, there was a clear increase in both forms of LC3B expression but the LC3B-I was significantly decreased with higher concentration of MPL (25 µM). Furthermore, MPL-induced autophagy was accompanied by enhanced accumulation of SQSTM1/p62 (Figure 4.9). These results imply that MPL-induced autophagy in OVCAR-3 and A2780 cell lines is SQSTM1/p62 dependent. Page 133 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.9: MPL regulates autophagy related proteins Western blot of lysates prepared from OVCAR-3 and A2780 cells treated with MPL (0, 10 and 25 µM) for the indicated period of time (48-72 h), were analysed for detection of autophagy markers (LC3B, Beclin 1 and SQSTM1/p62). The housekeeping gene (GAPDH) was used to confirm similar protein loading and blot transfer. Experiment repeated 2 times with similar results. A representative from three independent experiments is shown, n=3. Page 134 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.10 Inhibition of MPL-induced autophagy with 3-MA enhanced cell death Our results so far suggest that MPL induces autophagy. The induction of autophagy in response to metabolic and therapeutic stresses can have a pro- death or a pro-survival role, which contributes to the anticancer efficacy. To evaluate whether MPL-induced autophagy promotes cell death or survival, we applied pharmacologic inhibition of autophagy. 3-methyladenine (3-MA) is known to block autophagy at an early stage based on their inhibitory effect on class III PI3K activity (Castino, Bellio, Follo, Murphy, & Isidoro, 2010). 3-MA has a transient and short period of time effect (Wu et al., 2010). Figure 4.10 shows that in A2780 pre-treatment with 3-MA potentiated MPL- induced cell death compared with MPL or 3-MA treatment alone (cell proliferation dropped from 66.4 ± 2.26% to 49.6 ± 3.9% (P= 0.001) with 10 µM MPL and from 28 ± 3.5% to 24 ± 3.3% observed with higher concentration, 25 µM MPL). 3-MA in combination with 10 µM MPL led to an increase in OVCAR-3 cell survival, from 33 ± 1.3% to 51 ± 1.9% (P<0.0001). However, in the presence of 3-MA higher concentration of MPL (25 µM) cell death increased (percentage of cell proliferation decreased from 18 ± 1.4 to 12.7 ± 0.2, P=0.0048). Page 135 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.10: Inhibition of MPL-induced autophagy with 3-MA enhanced cell death OVCAR-3 and A2780 cells pre-treated with 3- methyl adenine (3-MA, class III PI3K inhibitor, 0.5 mM), 30 min. prior to treatment with indicated concentrations of MPL under the cell culture conditions. Proliferation was assessed using SRB assay. Data represent mean ± SEM, n=3. Page 136 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.11 Inhibition of MPL-induced autophagy with wortmannin enhanced cell death in OVCAR-3 To evaluate whether MPL-induced autophagy promotes cell death or survival, we applied pharmacologic inhibition of autophagy. Wortmannin are known as autophagy blockers at an early stage based on its inhibitory effect on class III PI3K activity (Castino et al., 2010). Wortmannin as an early stage autophagy inhibitor has persistent effects (Wu et al., 2010). Figure 4.11 shows that pre-treatment with wortmannin has no additional effect on 25 µM MPL, but reduced the percentage of cell proliferation from 55.41 ± 2.01 to 46.73 ± 2.12, P=0.0086, with 10 µM MPL in OVCAR-3. Page 137 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.11: Inhibition of MPL-induced autophagy with wortmannin enhanced cell death in OVCAR-3 OVCAR-3 cells pre-treated with Wortmannin (selective and irreversible inhibitor of PI3K, 100 nM), 30 min. prior to treatment with indicated concentrations of MPL under the cell culture conditions. Proliferation was assessed using SRB assay. Data represent mean ± SEM, n=3. Page 138 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.12 Morphological study of inhibition MPL-induced autophagy with 3- MA In the previous section, we illustrated that co-treatment with 3-MA potentiated cell death induced by MPL treatment. For more confirmation these results, ovarian cancer OVCAR-3 and A2780 cells were employed for this study. Cells were treated with MPL following pre-treatment with 3-MA for 72 h. The morphological changes are consistent with the previous data and results. Figure 4.12 presents pre-treatment with 3-MA resulted in amplified accumulation of autophagic marker (vacuoles) and dead bodies. Page 139 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.12: Morphological study of inhibition MPL-induced autophagy with 3-MA Cells were grown in 6 well tissue culture plates under standard cell culture conditions, pre-treated (30 min) with 3-MA (0.5 mM) followed by addition of MPL (0, 10, 25 µM) for 72 h. Cells were then stained with Giemsa (10% in PBS), washed and photographed under Leica DM IRB light microscope (magnification 40x). A representative from two independent experiments is shown, n=2. Page 140 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.13 Inhibition of MPL-induced autophagy with CQ has no extra effect on cell death To distinguish whether the accumulation of autophagosomes are caused by increased production or alternatively through reduced clearance due to disruption of the autophagic flux, we examined MPL-stimulated autophagosome formation in the presence and absence of chloroquine (CQ). CQ is a specific inhibitor of the lysosomal proton pump that disrupts the autophagic flux by preventing the fusion of autophagosomes with lysosomes (Mizushima, Yoshimori, & Levine, 2010). Pre-treatment with CQ did not influence MPL- induced cytotoxicity, suggesting that MPL-induced accumulation of autophagosomes were probably the consequence of increased production rather than any alteration clearance (Figures 4.13). Page 141 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.13: Inhibition of MPL-induced autophagy with CQ has no extra effect on cell death OVCAR-3 and A2780 cells pre-treated with chloroquine (CQ, lysosomal inhibitors, 2.5 µM), 30 min. prior to treatment with indicated concentrations of MPL under the cell culture conditions. Proliferation was assessed using SRB assay. Data represent mean ± SEM, n=3. Page 142 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.14 Morphological study of inhibition MPL-induced autophagy with CQ In the previous sections we illustrated that co-treatment with CQ has no extra effect on cell death induced by MPL treatment. To confirm these results, ovarian cancer OVCAR-3 and A2780 cells were employed for this study. Cells were treated with MPL following pre-treatment with CQ as a specific inhibitor of the lysosomal proton pump that disrupts the autophagic flux by preventing the fusion of autophagosomes with lysosomes, for 72 h. The morphological changes are consistent with the previous data and results. Figure 4.14 presents pre-treatment with CQ resulted in amplified accumulation of autophagic marker (vacuoles). Page 143 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.14: Morphological study of inhibition of MPL-induced autophagy with CQ Cells were grown in 6 well tissue culture plates under standard cell culture conditions, pre-treated (30 min) with CQ (2.5 µM) followed by addition of MPL (0, 10, 25 µM) for 72 h. Cells were then stained with Giemsa (10% in PBS), washed and photographed under Leica DM IRB light microscope (magnification 40x). A representative from two independent experiments is shown, n=2. Page 144 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.15 mTOR inhibitors sensitize ovarian cancer cells to MPL-induced cell death To investigate whether promotion of the autophagic pathway has an influence on MPL induced cell death, rapamycin and everolimus, potent inhibitors of mTOR, which has been shown to enhance autophagy (Moretti, Yang, Kim, & Lu, 2007), were used in our study. Co-treatment with rapamycin and everolimus suppressed A2780 cell proliferation further (the percentage of suppression in combination with rapamycin went down from 61.66 ± 1.3 to 33.72 ± 2.8 (P<0.0001) and with everolimus, from 73.50 ± 7.1 to 52.21 ± 3.5, P=0.044). However, in OVCAR-3 the combination of MPL with mTOR inhibitors had no suppressive effect on cell proliferation compared with either treatment alone (Figure 4-15). Page 145 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.15: mTOR inhibitors sensitize ovarian cancer cells to MPL- induced cell death Cells pre-treated with 50 nM rapamycin (Rap. mTOR inhibitor, autophagy inducer) and 1 nM everolimus (ROD001, mTOR inhibitor, autophagy inducer), 30 min. prior to treatment with MPL (10 µM) under the cell culture conditions. Data represent mean ± SEM, n=3. Page 146 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.16 Morphological study of the effect of mTOR inhibitors on cells treated with MPL In the previous sections we illustrated that co-treatment with mTOR inhibitors (rapamycin and RAD001 has no extra effect cell death induced by MPL treatment. For further confirmation of these results, ovarian cancer OVCAR-3 and A2780 cells were employed for this study. Cells were treated with MPL following pre-treatment with mTOR inhibitors as specific autophagy inducers by inhibiting mTOR, for 72 h. The morphological changes are consistent with the previous data and results. Figure 4.16 presents pre-treatment with these agents resulted in amplified accumulation of autophagic marker (vacuoles). Page 147 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.16: Morphological study of the effect of mTOR inhibitors on cells treated with MPL Cells were grown in 6 well tissue culture plates under standard cell culture conditions, pre-treated (30 min) with Rapamycin and RAD001 followed by MPL (0, 10, 25 µM) for 72 h. Cells were then stained with Giemsa(10% in PBS), washed and photographed under Leica DM IRB light microscope (magnification 40x). A representative from three independent experiments is shown, n=3. Page 148 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.17 MPL suppresses mTOR The mTOR/p70S6K signalling pathway is one of the well-established pathways negatively involves in the regulation of autophagy. mTOR associates with tumorigenesis and often activates in numerous types of tumors (Shintani & Klionsky, 2004). Therefore, we examined the effect of MPL on this pathway. To investigate whether mTOR was affected by MPL, we determined its cellular staining pattern using immuno-fluorescence confocal microscopy. Treated cells with MPL (0, 10 and 25 µM) after 4 h exhibited a markedly reduced level of punctate staining indicating the suppression of phosphorylated mTOR at Ser2448 (Figures 4.17). Page 149 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.17: MPL suppresses mTOR OVCAR-3 and A2780 were grown in 6 well tissue culture plates under standard cell culture conditions in the presence of MPL (0, 10, 25 µM) for 4 h. Cells were washed, fixed and incubated with primary antibodies in 1% BSA followed by related secondary antibodies in 1% BSA. Fluorescence images were observed and collected under Zeiss, Vert.A1, AxioCam, MRm (magnification 40x). A representative from two independent experiments is shown, n=2. Page 150 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.18 MPL inhibits mTOR downstream mediators, p70S6K and 4E-BP1 As we mentioned immuno-fluorescence confocal results indicated suppression of mTOR. For further proof, we employed western blot analysis to evaluate the expression of mTOR and its down-stream proteins. Western blot results illustrate that MPL decreased the expression of phosphorylated mTOR at Ser2448 and Raptor at Ser792, which is one of the mTORC1 complex members. If the mTOR is inhibited in these cell lines, then down-stream signalling molecules, p70S6K and 4E-BP1 are also likely to be affected. We therefore examined the expression of p70S6K and 4E-BP1 after treatment with MPL. MPL-treatment for up to 24 h resulted in complete inhibition of phosphorylated 4E-BP1 Thr37/46 in A2780, but only partial inhibition in OVCAR-3 cells. As shown in figure 4.18, the expression of mTOR target proteins, 4E-BP1 and p70S6K, were profoundly reduced in a time-dependent manner. Page 151 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.18: MPL inhibits mTOR downstream, p70S6K and 4E-BP1 Western blot of lysates prepared from OVCAR-3 and A2780 cells treated with 10 µM MPL for the indicated period of time (1, 4 and 24 h), were analysed for detection of mTOR, P70S6K, 4EBP1 and related phosphorylated proteins and p-Raptor. The house-keeping gene (GAPDH) was used to confirm similar protein loading and blot transfer. A representative from three independent experiments is shown, n=3. Page 152 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.2.19 MPL suppresses p70S6K expression Quantification of P-p70S6K revealed that in both cell lines inhibitory effects started after 1 h treatment with MPL. Percentage of inhibition in OVCAR-3 was 60.6 ± 0.54, P<0.0001 and in A2780, 56.38 ± 0.73, P=0.0007. Maximum inhibition was after 4 h of treatment. Compared to control, phosphorylation of p70S6K was suppressed by 33.87 ± 0.5%, P<0.0001 and 56.38 ± 0.73%, P<0.0001 in OVCAR-3 and A2780, respectively (Figure 4.19). These data confirm that suppression of the mTOR/p70S6K signalling pathway is involved in MPL- induced autophagy. Page 153 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 4.19: MPL suppresses p70S6K expression ELISA analysis was performed to quantify the level of Phospho-p70S6K at Thr389 and p70S6K in OVCAR-3 and A2780 cells after treatment (1, 4 and 24 h) with 10 µM MPL. Data represent mean ± SEM, n=3. Page 154 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 4.3 Discussion The most outstanding features of the data presented above supported the induction of autophagy followed by mTOR / p70S6K signalling pathway inhibition in human ovarian cancer cell lines, induced by the anthelmintic agent MPL. In Chapter 3, we had demonstrated that MPL, an anthelmintic compound, possesses potent anti-cancer activity in a range of cancer cells like ovarian cancer cells. Suppressive effects of MPL are implied by inhibiting cell proliferation, G1 cell cycle arrest and inducing cell death. Furthermore, our results indicated to MPL- induced cleavage of PARP-1. A more direct biochemical marker of apoptosis is the cleavage of PARP which revealed that treatment with the MPL induced significant cleavage (Berger, Berger, Catino, Petzold, & Robins, 1985). Therefore, we hypothesized that MPL might execute its anticancer activity by impeding the programmed cell death pathway, apoptosis. Apoptosis is executed by members of the caspase family of cysteine proteases, which can be activated by two main pathways: the extrinsic death receptor pathway and the intrinsic mitochondria/cytochrome c-mediated pathway (Estaquier, Vallette, Vayssiere, & Mignotte, 2012; Taylor, Cullen, & Martin, 2008). The two pathways linked, and both are trigger the activation of caspases 3, 8, and 9. In the death receptor pathway, extracellular death ligands bind to members of the tumour necrosis factor and nerve growth factor receptor superfamily to induce activation of caspase 8. Active caspase 8 in turn activates caspases 3 and 7, resulting in further caspase activation events and finally cell death (McIlwain, Berger, & Mak, 2013). Our results showed a clear contrast with no sign of activation of caspase-3 or caspase-8. The pan-caspase inhibitor z- VAD-fmk did not have any protective effect on the reduced viability in MPL- treated cells and furthermore, no significant differences in the percentage of annexin V-FITC positive cells and apoptosis related protein expression. Taken together, these results indicated that apoptosis might not play a role in MPL induced cell death in OVCAR-3 and A2780 cells. Page 155 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Here we noticed the occurrence of autophagy in MPL-exposed OVCAR-3 and A2780 cells, as proven by the increased number of acridine orange stained acidic autophagolysosomes and LC3-II accumulation. In addition to LC3, SQSTM1/p62 is another frequently used autophagy marker, which is linked LC3 and the ubiquitinated proteins during autophagy. As polyubiquitinated substrates are transferred into the intact autophagosome to degrade in autolysosomes, SQSTM1/p62 is also degraded with its ubiquitinated substrate. Therefore, SQSTM1/p62 is identified as a negative marker of autophagic degradation (Puissant, Fenouille, & Auberger, 2012), and the clearance of SQSTM1/p62 may be a positive indicator of complete autophagy (Lippai & Low, 2014). In our study, 10 µM MPL increased the expression of SQSTM1/p62 while it suppressed with higher concentration of MPL which is indicated to complete autophagy induced with 25 µM MPL (elevated LC3B-II along with degradation of other markers). Recently, many chemotherapeutic candidates have been reported to induce autophagy, but the roles of altered autophagy are inconsistent, because it can either protect cells from apoptosis, or promote cell death (Maes, Rubio, Garg, & Agostinis, 2013). Numerous reports suggest that autophagy is a survival mechanism protecting cells from cell death due to DNA damage. Other studies indicate that autophagy may be the mechanism of cell death during tumour treatment or that may be involved in the induction of apoptosis (Baehrecke, 2005; N. Chen & Karantza- Wadsworth, 2009). Pharmacological inhibition of autophagy could potentiate the effects of a compound and lead to cell death, thus it known as “protective autophagy”. While under some conditions, autophagy inhibition decreases the efficacy of chemotherapy and will protect the cell from death (Z. J. Yang, Chee, Huang, & Sinicrope, 2011). In this study, we found that pharmacological inhibition of autophagy in the early stage of this process, with 3-MA or wortmannin potentiates the anticancer activity of MPL, which raises this Page 156 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer suggestion that autophagy induced by 10 µM MPL is more protective in ovarian cancer cells, rather than suppressive. Notably, CQ as an end-stage autophagy inhibitor, by targeting lysosomal proton pump had no effect on the suppressive process of MPL. Having seen that MPL induces autophagy, we next need to clarify the mechanism behind it. Accumulated evidence supports the idea that mTOR is a best-known negative regulator for autophagy and it plays a variety of physiological roles, including the regulation of cell growth and cell survival (Altomare & Khaled, 2012; Neufeld, 2012). To find out whether MPL-induced autophagy mediated via mTOR / p70S6K, we combined rapamycin and everolimus as well-known mTOR inhibitors with MPL. Our results indicate that these inhibitors enhance chemo-sensitivity to MPL, and seem to do so through autophagy. We have shown that MPL might induce autophagy by involving mTOR pathways. In this study, we observed that p-mTOR was de-phosphorylated at Ser2448, which is one of the most critical residues for catalytic activity of mTOR. In addition to p-mTOR, MPL de-phosphorylated other important components of the mTORC1 complex such as p-Raptor in a time-dependent manner. This resulted in the efficient de-phosphorylation of the downstream substrates of mTOR, Raptor, p70S6K and 4EBP1. These observations show that MPL cytotoxicity to ovarian cancer cells is due to inhibition of the mTORC1 activity. Conclusion: In summary, our finding clearly demonstrated that MPL has suppressive activity on the mTOR / p70S6K signalling pathway, which resulted in the up-regulation of biological and morphological markers of autophagy and DNA damage. Furthermore, MPL treatment inhibited apoptosis by down-regulating caspase-3 and 8. Page 157 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Chapter 5: Preclinical study of antitumor effects of the monepantel (ADD-1566) against human ovarian cancer cells in a xenograft model 5.1 Aim We have shown in-vitro anti-proliferative activity of MPL (F. Bahrami, D. L. Morris, et al., 2014; F. Bahrami, Pourgholami, Mekkawy, Rufener, & Morris, 2014). Exposure to MPL prevents the proliferation of ovarian cancer cell lines (OVCAR-3 and A2780). To provide a further preclinical rationale for the development of this agent in clinic, we initiated the current pilot study, testing the effects of MPL in-vivo against human OVCAR-3 tumor growth in a nude xenograft model. Page 158 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 5.2 Results 5.2.1 MPL impacts on subcutaneous tumor To test the in vivo significance of our cellular observations, we determined the effect of MPL administration on OVCAR-3 xenograft growth in nude mice. MPL was administered by intraperitoneal injection (IP) 3 times a week at 25 and 50 mg/kg into animals bearing OVCAR-3 subcutaneous (SC) tumors. Fig. 5.1 clearly shows that MPL decreased tumor volumes compared to the control group and slowed down the relative tumor progression. After 3 weeks of treatment, the average tumor volume in treated mice with 25 and 50 mg/kg MPL were 206.28 ± 59.26 and 112 ± 18.14 mm3, respectively, which were about %25 and %60 lower than the average tumor volume in control mice (279.5-± 73.16 mm3 ). Consistent with the previous results, there were reduction in the average of tumor weight in treated mice with MPL in comparison with control mice (From 0.200 ± 0.036 g in control mice decreased to 0.183 ± 0.037 and 0.100 ± 0.0 in MPL treated mice in dose-dependent manner) (Figure 5.1). These results indicated that MPL significantly reduces growth of OVCAR-3 xenografts in nude mice without causing any observable side effects or weight lost. Page 159 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 5. 1 MPL reduces growth of ovarian cancer xenografts (A) Antitumor effects of MPL tested against subcutaneous xenografts in nude mice, OVCAR-3 cells (2.5×106) were injected SC into the flank of each athymic female nude mouse (n=15). Treatment with MPL administered IP, 3 times weekly at total daily concentration of 25 or 50 mg/kg/day were initiated on day 7 of cell inoculation (n= 5 mice / group). Sterile 0.5% HPMC was the vehicle for MPL, the control group thus received vehicle as treatment. Calliper measurement of tumor volumes were recorded 3 times every week. Mean±SEM from these measurements is plotted for each group. (B) MPL treatment did not affect animal weights. (C) Representative, tumor weight in nude mice at time of sacrifice (day 22 of study), (D) Representative, tumor size in nude mice at time of sacrifice (day 22 of study). Page 160 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 5.2.2 MPL induces necrosis of ovarian malignancy in-vivo To verify and assess the activity of MPL to induce necrosis, in-vivo, we next employed hematoxylin and eosin staining immunohistochemical analysis. Histological evaluation revealed percentage of necrosis were from 8.5 ± 1.3 in animals with no treatment to 10.5 ± 2.08 and 21.5 ± 7.75 in 25 and 50 mg/kg MPL treated mince, respectively. Therefore, analysis of tumors’ images indicating profound drug-induced necrosis, which is concentration-dependent. Page 161 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 5. 2 MPL induces necrosis of ovarian malignancy, in-vivo OVCAR-3 cells (2.5 × 106) were injected SC into the flank of each athymic female nude mouse (n=15). Treatment with MPL administered IP, 3 times weekly at a total daily concentration of 25 or 50 mg/kg/day were initiated on day 7 of cell inoculation (n=5 mice / group). With sterile 0.5% HPMC being the vehicle for MPL, the control group thus received vehicle as treatment. Histological images of H&E stained tumors shown on top, indicating profound drug-induced necrosis (black arrow head; magnification, 10x). Analysis of necrosis was performed based on number of the field / section, section = 5, % of necrosis = necrosis area / Total area × 100. Page 162 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 5.2.3 MPL inhibits mTOR / p70S6K signalling pathway The ability of MPL to inhibit mTOR and its pathways in ovarian cancer cells in-vitro was a good indication that it has the potential to affect the mTOR levels in-vivo. To confirm the in-vitro findings, we looked at mTOR signalling pathway and related protein expression. Western blot results indicated that MPL decreased the expression of phosphorylated mTOR at Ser2448, in-vivo. If mTOR is inhibited in the tumor sample, then down-stream signalling molecules, p70S6K and 4E-BP1 are also likely to be affected. We therefore examined the expression of p70S6K and 4E-BP1, on tumor lysate. IP treatment with MPL resulted in complete inhibition of phosphorylated P-p70S6K Thr389 and 4E-BP1 Thr37/46. Thus, as shown in figure 5.3, the expression of mTOR target proteins, 4E-BP1 and p70S6K, were highly reduced in a dose-dependent manner after MPL administration. Page 163 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 5. 3 MPL inhibits mTOR / p70S6K signalling pathway OVCAR-3 cells (2.5 × 106) were injected SC into the flank of each athymic female nude mouse (n=15). Treatment with MPL administered IP, 3 times weekly at total daily concentration of 25 or 50 mg/kg/day were initiated on day 7 of cell inoculation(n= 5 mice / group). Sterile 0.5% HPMC was the vehicle for MPL, the control group thus received vehicle as treatment. Western blot of lysates prepared from MPL treated / no treated SC OVCAR-3 tumors. Blots were analysed for detection of mTOR, P70S6K, 4EBP1 and related phosphorylated proteins. The housekeeping gene (GAPDH) used to confirm similar protein loading and blot transfer. Page 164 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 5.2.4 MPL affects cell cycle regulators To confirm the cell cycle arrest followed by mTOR signalling inhibition through MPL treatment, we looked at cell cycle regulator proteins notably cyclins D1 and E2 together with their associated cyclin-dependent kinases CDK2 and CDK4 (Figure 5.4). Diminished cell cycle regulators proteins, like cyclin D1 and E and related CDK2 and CDK4, accompanied MPL-induced G1 cell cycle arrest. These results further confirm the induction of G1 cell cycle arrest after MPL inhibition of mTOR / p70S6K signalling. Page 165 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 5. 4 MPL affects cell cycle regulators OVCAR-3 cells (2.5 × 106) were injected SC into the flank of each athymic female nude mouse (n=15). Treatment with MPL administered IP, 3 times weekly at total daily concentration of 25 or 50 mg/kg/day were initiated on day 7 of cell inoculation (n=5 mice / group). Sterile 0.5% HPMC being the vehicle for MPL, the control group thus received vehicle as treatment. Western blot of lysates prepared from MPL treated / untreated SC OVCAR-3 tumors. Blots were analysed for detection of Cyclin D1, E2 and CDK 2 and 4 proteins. The housekeeping gene (GAPDH) was used to confirm similar protein loading and blot transfer. Page 166 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 5.2.5 MPL induces cell cycle arrest through inhibition retinoblastoma protein (Rb) It has reported that Rb can actively inhibit cell cycle progression when it is dephosphorylated (Jeong, Hong, Jeong, & Koo, 2011). To determine whether the cell cycle arrest by MPL was mediated through suppressing the phosphorylation of Rb, we detected the expression of Rb and its related phosphorylated form measured by western blot. As shown in Fig. 5.5, compared with the vehicle treated group, treatment with MPL resulted in suppression of Rb. These results indicate that the cell cycle distribution by MPL results from suppressing the phosphorylation of Rb. Page 167 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Figure 5. 5 MPL induces cell cycle arrest through inhibition retinoblastoma protein (Rb) OVCAR-3 cells (2.5 × 106) were injected SC into the flank of each athymic female nude mouse (n=15). Treatment with MPL administered IP, 3 times weekly at total daily concentration of 25 or 50 mg/kg/day were initiated on day 7 of cell inoculation (n=5 mice / group). Sterile 0.5% HPMC was the vehicle for MPL, the control group thus received vehicle as treatment. Western blot of lysates prepared from MPL treated / untreated SC OVCAR-3 tumors. Blots were analysed for detection of retinoblastoma proteins (Rb) and its phosphorylated. The housekeeping gene (GAPDH) was used to confirm similar protein loading and blot transfer. Page 168 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 5.3 Discussion Based on the accumulated data in previous chapters we provided proof of concepts of suppressive effects of MPL on variety of cancer cell lines, especially ovarian malignancy. Consistent with our previous results; MPL induces cell death through G1 cell cycle arrest. While MPL has the ability to cleave PARP-1, it inhibits apoptosis through down-regulation caspase-3 and 8. In addition to inhibition of apoptosis, our data supported another role for MPL, induction of autophagy, via increasing acidic vacuoles and LC3 shift. Notwithstanding, MPL-induced autophagy was through inhibition of mTOR/ p70S6K pathway as a major checkpoint in signalling pathway regulating autophagy. To explore the proof of concepts of in-vitro findings, we planned a pilot in-vivo experiment with the aim of determination the dose-response activity and safety of MPL in nude mice bearing SC xenograft of human ovarian epithelial cancers, OVCAR-3. We already know from publications by Novartis and European drug regulatory organizations (Dadak, Asanger, Tichy, & Franz, 2013; Jones et al., 2010; Malikides et al., 2009), that MPL given oral, SC and iv are well tolerated in sheep, cattle, rats and mice. Repeated dose (GLD) toxicity study over 3 months in mice with 10 animals per dose per sex, has previously examined dose of 1000 mg/kg/bw/day. No treatment-related clinical signs of toxicity and no significant differences in body weight or food consumption were observed. Thus, based on reports, at doses stated above, and far over, no adverse effects were seen in mice treated. In this investigation, mice will be female nude 6 weeks old, due to most commonly studied and used in this type of investigation known biology and Page 169 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer behaviour, easy to handle. We follow planned schedules as the following flow diagram: Figure 5. 6 Flow diagram summarizing in-vivo experiments The scope of the research include the investigation of the in-vivo efficacy against proliferation and metastasis of MPL using animal models; to this end, our studies have shown that MPL has in-vivo suppressive efficacies against OVCAR-3 tumour using a xenograft mouse model Our data shows that in MPL treated mice, tumour volumes are significantly lower than the control group, with no observed adverse effect in both control and treated group, which are in line with Novartis reports (Malikides et al., 2009). The in-vivo data correlated with increased level of necrosis. Page 170 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Another observation that we report is the reduction of tumour volume size by MPL as demonstrated by significantly fewer and smaller tumour volume in the treated mice compared with control group. Because activation of mTOR / p70S6K pathway is associated with the regulation of cell growth, protein translation, metabolism, cell invasion, and cell cycle (Vignot, Faivre, Aguirre, & Raymond, 2005). Therefor, attenuation of tumours seen in our study could be due to the inhibition of mTOR pathway by MPL. Major downstream targets of mTOR are p70S6K and 4E-BP1, which are activated by mTOR and then dissociate from the eukaryotic translation factor (eIF-4E) and activates protein synthesis (Kapoor, 2009). Overexpression or over activation of mTOR may strengthen the signals passed down by mTOR signalling pathway, which will cause over-phosphorylation of the downstream molecules p70S6K and 4E-BP1. Once phosphorylated, p70S6K and 4E-BP1 can promote protein synthesis (Li et al., 2011). Thus, several cell-cycle related proteins including cyclin D1, cyclin E, will be excessively upregulated, which resulted in the progression of cell cycle. We found that the expression of p- p70S6K, p-4E-BP1, cyclin D1, cyclin E2 are down regulated in MPL treated mouse, consistent with our previous in-vitro chapter. Therefore, MPL by inhibition of mTOR pathway provides tumour with G1 cell cycle arrest following by halting tumour growth advantage. The retinoblastoma protein (Rb) is an important tumour suppressor. It is a key to regulate cell cycle in a phosphorylation-dependent manner. Cyclin D, partnered with either CDK4 or CDK6, phosphorylates the Rb to generate what has been called hypophosphorylated Rb (Foster, Yellen, Xu, & Saqcena, 2010; Narasimha et al., 2014). Our data showed that the expression of phosphorated retinoblastoma protein (p-Rb) decreased in dose-related manner which consequence of cyclin D and related CDKs down regulation. Page 171 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Conclusion: In summary, our results provide further support for the suppressive potential of MPL treatment in the ovarian cancer tumours, in-vivo. MPL demonstrated a dose-dependent inhibitory effect in OVCAR-3 xenograft model and was relatively nontoxic at effective doses, along with G1 cell cycle arrest via inhibition mTOR / p70S6K signalling pathway. Page 172 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Chapter 6: General Discussion and Future Direction Our laboratory focused on discovering improved treatments for epithelial ovarian cancer. Ovarian cancer has a high healthcare burden due to low cure rates and frequent recurrent disease that causes significant symptoms for patients. This is despite the fact that ovarian cancer is initially sensitive to systematic treatments and most patients are free of disease after completing initial surgery and chemotherapy. The fundamental problem that we are addressing is to understand how ovarian cancer cells escape initial treatment and the aim of the pharmacology and drug development are to optimise the pre- clinical development and science-led clinical application of novel therapies, including “first into man” (phase I) studies. Aim of this work was to evaluate the potential anti-cancer activity of an AAD compound, MPL as an anthelmintic agent and to determine its mechanism of action. MPL is used against nematodes and is known as a safe agent and selectively act as an agonist on an acetylcholine nicotinic receptor related structure designated ACR-23/MPTL-1 (Rufener et al., 2013). Here we present the first report to show that MPL significantly inhibits proliferation and can suppress tumour development in ovarian cancer cell lines under in-vitro cell culture and in-vivo conditions, and possible involved mechanism behind it. To find out if the MPL effect is selective towards malignant cells, a number of normal cells including HOSE, CHO, HUVEC and HEK were also treated with MPL. Comparisons of the IC50 values indicate that, MPL is approximately 10 times more toxic to the ovarian cancer cells than it is to the normal human epithelial cells (HOSE). In line with in-vitro findings, experiments conducted on tumor bearing mice demonstrated dose-dependent suppression of SC tumour Page 173 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer growth by MPL. Because of MPL treatment, tumour volumes in the drug treated groups were profoundly lower than the control group. Consequently, it took a considerably longer time for their tumours to reach the limit volume of 1000 mm3. MPL treatment did not cause any observable adverse effect in these animals. These results are consistent with literature showing the exceptional safety of MPL in in-vitro as well as animals (Hosking et al., 2009; Skripsky & Hoffmann, 2010). The anthelmintic activity of MPL has been related to its effects on the nematode nAChR α-subunit analogue that holds resemblance to the second transmembrane domain of nAChRα7 subunit and studies on C. elegans indicate that a choline-activated ion channel which is known to be partially permeable to Ca2+mightbe involved in the MPL mediated nematicidal activity (Treinin et al., 1998; Yassin et al., 2001). Here in our study, we found that neither acetylcholine nicotinic receptor antagonist (including the selective α7-nAChRs antagonist and α-bungarotoxin (Donnelly-Roberts & Lentz, 1991) have the capacity to prevent the antiproliferative efficacy of MPL in epithelial ovarian cancer cells. These observations suggest that at least in these cells, MPL- induced cytotoxicity is not mediated via nAChRs. Subsequent experiments revealed MPL-induced G1 cell cycle arrest in cancer cells in-vitro which was in line and consistent with in-vivo findings. Inhibition of cellular thymidine incorporation followed by the cleavage of PARP-1 confirmed that MPL inhibits growth and proliferation of cancer cells. At this stage, these observations can perhaps be best explained by the effect of MPL on the cell cycle where progression from the G1 phase is halted. Cancer is one of several diseases considered a cell cycle related phenomenon. In the normal cell, the transition from one phase to another occurs in an orderly fashion well regulated by various proteins. The cell cycle is tightly controlled at specific points by CDKs, which play a crucial role in cell cycle progression. Of the various CDKs identified so far, CDK2 and CDK4 seem essential for entry in G1 Page 174 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer and G1-S transition (Tanaka & Araki, 2010). For activation, the CDKs require different cyclins at different phases of the cycle. For the G1 transition and progression of the cell cycle, cyclins A, D and E are required (Baldin, Lukas, Marcote, Pagano, & Draetta, 1993). Cyclins A and E bind to CDK2 while cyclin D1 binds to CDK4 and CDK6 (Vermeulen, Van Bockstaele, & Berneman, 2003). As evidenced by increased percentage of cells in the G1 phase accompanied by the sharp decline of cells present in the S phase, it is obvious that in these epithelial ovarian cancer cells, MPL induces cell cycle arrest at the G1 phase. This is confirmed by depressed expression of essential cell cycle regulatory proteins cyclin D1 and cyclin A and their CDKs, CDK4 and CDK2 respectively. However, the cyclin E2 results are not so obvious. Whereas in OVCAR-3 cells there was no detectable change in E2 expression with increasing MPL concentrations, in A2780 cells which normally express low levels of E2, a profound increase in cyclin E2 expression is seen at the highest MPL concentration used (25 µM). This reveals the very complex nature of the interaction between the cell cycle regulatory apparatus. Based on previous reports on the interaction of the cyclins and their kinases in ovarian cancer, it seems that while both D1 and E2 cyclins act as positive regulators, suppression of cyclin D1 expression drives the cell to produce more of the downstream cyclin E2 to compensate for the D1 loss. However, it has been shown that, suppression of cyclin D1 can still cause G1 cell cycle arrest. Bowe and colleagues have shown that in mice with growing mammary tumors, cyclin D1 deficiency is compensated by cyclin E2 expression (Bowe et al., 2002). Masamha and Benbrook have elegantly demonstrated that in ovarian cancer cells irrespective of p53 status, loss of D1 and increased cyclin E2 expression still cause cell cycle arrest at the G1 phase. Thus suggesting that in ovarian cancer cells, attenuation of cyclin D1 availability is sufficient to induce G1 cell cycle arrest (Masamha & Benbrook, 2009). Additionally, consistent with the inhibition of cell proliferation, cyclin A levels, which positively correlate with cell proliferation (Khan et al., 2003) were reduced in MPL treated cells. CDK inhibitors such as p27Kip1 negatively regulate the cell cycle progression. Thus, increased cellular CDK inhibitor levels contribute towards G1 cell cycle arrest Page 175 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer (Shapiro, 2006). In MPL treated cells, up-regulation of p27Kip1 observed. Moreover, in the current study, we investigated cell cycle distribution in tumours derived from OVCAR-3 cells subcutaneously implanted in nude mice. Many studies have shown an association between cell cycle and cancer and inhibition of the cell cycle has considered as a target for the management of cancer (Pohl et al., 2003). We have demonstrated for the first time that MPL induces G1 cell cycle arrest in tumour cells, in-vivo in parallel to our findings in-vitro. Our results demonstrated that, MPL blocked cell cycle progression at the G1 phase, which correlated with a remarkable decrease in the expression of cyclin D1 and phosphorylation of pRb, two major cell-cycle regulators. Cell cycle arrest accordingly causes a reduction in lower thymidine uptake and DNA synthesis. Depending on various other factors, events associated with cell cycle arrest can lead to cleavage of PARP. PARP expression is frequently up regulated in ovarian serous carcinomas and may serve as a marker of aggressive behaviour with prognostic value (Brustmann, 2007). It is well established that, PARP help cells to maintain their viability and hence cleavage of PARP facilitates cellular disassembly and serves as marker of cells undergoing cell death as its cleavage prevents survival (Bowe et al., 2002). Our results show that MPL exerts time and concentration-dependent cleavage of PARP-1. Western blot analysis of MPL-treated cells suggests that the A2780 cells are probably more sensitive to the MPL-induced PARP cleavage than OVCAR-3 cells. This may be partly related to the wild-type p53 status of A2780 cells compared to mutated expression of p53 in OVCAR-3 cells. PARP-1 regulates the stability of the wild type p53 protein (Wesierska-Gadek & Schmid, 2001). Furthermore, p53 can regulate both necrotic and apoptotic cell death. Therefore, mutations or deletions in this tumour-suppressor protein may be selected by cancer cells to provide not only their resistance to apoptosis but also to necrosis, and explain resistance to chemotherapy and radiation even when it kills via non-apoptotic mechanisms (Montero, Dutta, van Bodegom, Weinstock, & Letai, 2013). Page 176 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer In line with our finding on MPL-induced cleavage of PARP-1, and the association of this marker with apoptosis(F. Bahrami, D. L. Morris, et al., 2014)(Bahrami et al., 2014b), we anticipated that MPL may act as an apoptogenic agent. Published literature is divided as to whether PARP-1 cleavage is an event that precedes apoptotic cell death or is a marker of another distinct mechanism of cell death (Chaitanya et al., 2010). Our results however, did not show caspase-3 or caspase-8 activation in MPL-treated cells. Apoptotic features such as morphological changes in favour of apoptosis, increased level of Annexin-V+ or DNA fragmentation, were not detected. Additionally, the MPL-induced antiproliferative effect was not prevented by pre- treatment with the pan-caspase inhibitor z-VAD-fmk, thus further confirming the induction of cell death is independent of the caspase-mediated apoptotic pathways. These results collectively suggest that cell death induced by MPL in ovarian cancer cell lines is not an apoptotic-mediated event. Instead, we conclusively found that cells treated with MPL undergo autophagy. Through LC3B translocation studies, we were able to find that MPL treated cells present with typical autophagic morphology and biochemical signature. The autophagic effect of MPL was evident through drug-induced expression of SQSTM1/p62 together with the conversion of LC3B-I to LC3B-II in a time and concentration dependent manner. SQSTM1/p62 protein interacts with LC3B-II (Bauvy, Meijer, & Codogno, 2009; Mizushima & Yoshimori, 2007) and is degraded in autophagolysosomes. Therefore, its reduction indicates increased autophagic degradation, whereas an increase of SQSTM1/p62 indicates incomplete autophagy (Bjorkoy et al., 2009). On this line of thought, through accumulation of SQSTM1/p62 and LC3B-II, 10 µM MPL induces incomplete and non-productive autophagy while, higher concentration of MPL (25 µM) triggers active and complete autophagy (elevated LC3B-II along with degradation of other markers). The concept of “autophagic cell death” is commonly accepted based on the presence of autophagic features in dying cells and cell survival via suppression of autophagy (Maiuri, Zalckvar, Kimchi, & Page 177 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Kroemer, 2007; Scarlatti, Granata, Meijer, & Codogno, 2009). Additionally, because autophagy may play a role as a cell survival pathway in response to therapeutic agents the induction of autophagy may work in favour of cancer cells (Rouschop & Wouters, 2009). Cell rescue experiments in which OVCAR-3 and A2780 cells were pre-treated with autophagy inhibitors as 3-MA resulted in decreasing cell viability compared with cells treated with MPL alone. Similar results were obtained in OVCAR-3 from experiments using wortmannin, an agent that inhibits the autophagic process at early stage (Wu et al., 2010). Moreover, the specific end-stage autophagy inhibitor, CQ targeting the lysosomal proton pump did not influence the antiproliferative effect of MPL or the drug-induced morphology changes. The potentiated cell death through pre- treatment with autophagy inhibitors in cells treated with MPL indicates that the autophagy may serve as an additional target as pro-survival and the other mechanisms of cell death may be involved in MPL-induced cytotoxicity. Autophagy is mainly regulated through the mTOR / p70S6K as an important signalling pathway and for its ability to halt and regulate cellular catabolic processes, which is frequently deregulated in cancer and metabolic disorders in eukaryotic cells. This pathway also play a variety of physiological roles, for instance as a positive regulator of protein synthesis, cell growth and proliferation. mTOR / p70S6K signalling is also known for (Banerjee, Beal, & Thomas, 2010; Nixon, 2013; Sridharan, Jain, & Basu, 2011). Consistent with this, our data presented the negative effect of MPL on the mTOR / p70S6K pathway. Combined use of mTOR inhibitors (rapamycin and RAD001) and MPL resulted in further suppression of cell viability, suggesting that the inhibition of mTOR signalling may play a role in MPL-induced autophagy. We found that MPL markedly inhibits mTOR phosphorylation at Ser2448, which then prevents activation of the mTOR signalling. On this basis, inhibition of p70S6K and 4E- BP1 may be directly resulting from their upstream, inhibition of mTOR by MPL. Consistent with these results and as proof of concept to our in-vitro findings, MPL reduced the expression of mTOR phosphorylation at Ser2448 in OVCAR-3 Page 178 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer xenograft mice. Accumulated studies shows that deregulation of mTOR signalling are usually related to tumour growth and angiogenesis via 4E-BP-1 and p70S6K (Li et al., 2011; Peponi et al., 2006). The mTOR inhibitors exhibited long-acting tumour suppression in clinical trial (Xu et al., 2014). In this in-vivo study, we clearly demonstrated, MPL reduces tumour size and decrease in the level of phosphorylated 4E-BP1 and p70S6K. By inhibiting mTOR pathway, MPL suppresses ovarian cancer cells viability and tumour growth via inhibiting cyclin D1 and CDK4 activity which leading to Rb hypo-phosphorylation and cell cycle arrest. Upon phosphorylation, pRb releases the transcription factor E2F, which activates the transcription of genes required for G1/S phase transition (Matsushime et al., 1994). Thus and In addition to its effect on cyclin D1, MPL strongly inhibits the phosphorylation of pRb in xenograft OVCAR-3 model. Conclusion: In conclusion, the present study used a cell culture system (in-vitro) and a tumor-xenograft mouse model (in-vivo) to demonstrate for the first time that MPL effectively inhibits ovarian cancer cell proliferation and tumor growth. These results provide an explanation for the anti-tumour activity of MPL. The pharmacologic significance of MPL-induced inhibition of mTOR leads to vacuole formation and autophagy as well as G1 cell cycle arrest and inhibition of protein synthesis. This is justified by the solid evidence showing inhibition of apoptosis followed by the induction of autophagy. Taken together these observations explain the possible mechanism behind the inhibition of growth and proliferation seen in MPL-treated cells. We have shown that MPL modulates cell cycle regulatory proteins D1, A and E2, reduces the expression of associated cyclin-dependent kinases CDK2 and Page 179 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer CDK4 accompanied by up-regulation of p27Kip1 culminating in G1 cell cycle arrest and cleavage of PARP-1, while inhibiting apoptosis. Moreover, our studies provide the general mechanism behind the MPL- induced autophagy in ovarian cancer cells. We provide proof-of-concept that MPL induces autophagy by suppressing mTOR/p70S6K pathway (Figure 6.1). Thus, our data provide evidence that PI3K/mTOR pathway suppression could be a potential mechanism for MPL induced autophagy. Furthermore, and to complete our investigation, MPL demonstrated a dose- dependent tumor suppression effect in an ovarian cancer xenograft model and was relatively nontoxic to other normal tissues and organs at its effective doses. Our study demonstrates that MPL effectively inhibits human ovarian cancer cell proliferation and tumor growth in vivo as well as in vitro, possibly by inducing G1 cell cycle arrest and deactivating mTOR pathway. These results suggest that MPL could be a potential candidate for the development of novel treatment strategies for human ovarian malignancies. Figure 6. 1 The proposed mechanism of MPL-induced autophagy through mTOR / p70S6K- 4E-BP1 inhibition Page 180 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Future Directions: In this study, we have characterized the underlying mechanism of MPL mediated mTOR inhibition and induction of autophagy in OVCAR-3 and A2780 cells as potential anti-cancer agent. However, there are still number of issues, which need to clarify: - We provided data to show that MPL inhibited apoptosis. Beside that autophagy was introduced as pro-survival agents. So; what is the main reason of cell death induced by MPL? Clarify the mechanism involve cell death induced by MPL. - If MPL-induced autophagy is pro-survival, what will be the consequences of pharmacological or biological inhibition of autophagy? We should fogous more on silencing the autophagy regulators and applying another potential anticancer or autophagy inhibitor agents in combine with MPL for more investigation. - Accumulated data presented to support the suppressive effect of MPL on mTOR / p70S6K pathway. We should clarify whether it is mTOR inhibitor or this action is consequences of up-stream regulators inhibition. Thus, need further investigation on the possible MPL interaction with up-stream regulator of mTOR pathway and find out the complete mechanism behind the mTOR inhibition. - In future, we need more pre-clinical xenograft models to investigate about dose – response and more investigation about possible suppressive effect on tumour growth, anti-angiogenesis and anti- metastasis effects of MPL, alone or in combination with other agents (for instance, taxol, tetracyclines etc.) and / or with other different ovarian cancer cell lines. Page 181 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer References Abraham, R. T. (2004). PI 3-kinase related kinases: 'big' players in stress- induced signaling pathways. DNA Repair (Amst), 3(8-9), 883-887. doi: 10.1016/j.dnarep.2004.04.002 Al Rawahi, T., Lopes, A. D., Bristow, R. E., Bryant, A., Elattar, A., Chattopadhyay, S., & Galaal, K. (2013). Surgical cytoreduction for recurrent epithelial ovarian cancer. Cochrane Database Syst Rev, 2, CD008765. doi: 10.1002/14651858.CD008765.pub3 Albert, J. M., Kim, K. W., Cao, C., & Lu, B. (2006). Targeting the Akt/mammalian target of rapamycin pathway for radiosensitization of breast cancer. Mol Cancer Ther, 5(5), 1183-1189. doi: 10.1158/1535- 7163.MCT-05-0400 Altomare, D. A., & Khaled, A. R. (2012). Homeostasis and the importance for a balance between AKT/mTOR activity and intracellular signaling. Curr Med Chem, 19(22), 3748-3762. Amaravadi, R. K., Yu, D., Lum, J. J., Bui, T., Christophorou, M. A., Evan, G. I., . . . Thompson, C. B. (2007). Autophagy inhibition enhances therapy- induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest, 117(2), 326-336. doi: 10.1172/JCI28833 Averous, J., Fonseca, B. D., & Proud, C. G. (2008). Regulation of cyclin D1 expression by mTORC1 signaling requires eukaryotic initiation factor 4E- binding protein 1. Oncogene, 27(8), 1106-1113. doi: 10.1038/sj.onc.1210715 Baehrecke, E. H. (2005). Autophagy: dual roles in life and death? Nat Rev Mol Cell Biol, 6(6), 505-510. doi: 10.1038/nrm1666 Page 182 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Bahrami, B. F., Ataie-Kachoie, P., Pourgholami, M. H., & Morris, D. L. (2014). p70 Ribosomal protein S6 kinase (Rps6kb1): an update. J Clin Pathol. doi: 10.1136/jclinpath-2014-202560 Bahrami, F., Morris, D. L., Rufener, L., & Pourgholami, M. H. (2014). Anticancer properties of novel aminoacetonitrile derivative monepantel (ADD 1566) in pre-clinical models of human ovarian cancer. Am J Cancer Res, 4(5), 545-557. Bahrami, F., Pourgholami, M. H., Mekkawy, A. H., Rufener, L., & Morris, D. L. (2014). Monepantel induces autophagy in human ovarian cancer cells through disruption of the mTOR/p70S6K signalling pathway. Am J Cancer Res, 4(5), 558-571. Bai, X., & Jiang, Y. (2010). Key factors in mTOR regulation. Cell Mol Life Sci, 67(2), 239-253. doi: 10.1007/s00018-009-0163-7 Baldin, V., Lukas, J., Marcote, M. J., Pagano, M., & Draetta, G. (1993). Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev, 7(5), 812-821. Banerjee, R., Beal, M. F., & Thomas, B. (2010). Autophagy in neurodegenerative disorders: pathogenic roles and therapeutic implications. Trends Neurosci, 33(12), 541-549. doi: 10.1016/j.tins.2010.09.001 Barth, S., Glick, D., & Macleod, K. F. (2010). Autophagy: assays and artifacts. J Pathol, 221(2), 117-124. doi: 10.1002/path.2694 Bast, R. C., Jr., Hennessy, B., & Mills, G. B. (2009). The biology of ovarian cancer: new opportunities for translation. Nat Rev Cancer, 9(6), 415-428. doi: 10.1038/nrc2644 Bauvy, C., Meijer, A. J., & Codogno, P. (2009). Assaying of autophagic protein degradation. Methods Enzymol, 452, 47-61. doi: 10.1016/S0076- 6879(08)03604-5 Ben-Ami, H. C., Yassin, L., Farah, H., Michaeli, A., Eshel, M., & Treinin, M. (2005). RIC-3 affects properties and quantity of nicotinic acetylcholine Page 183 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer receptors via a mechanism that does not require the coiled-coil domains. J Biol Chem, 280(30), 28053-28060. doi: 10.1074/jbc.M504369200 Benjamin, D., Colombi, M., Moroni, C., & Hall, M. N. (2011). Rapamycin passes the torch: a new generation of mTOR inhibitors. Nat Rev Drug Discov, 10(11), 868-880. doi: 10.1038/nrd3531 Berger, N. A., Berger, S. J., Catino, D. M., Petzold, S. J., & Robins, R. K. (1985). Modulation of nicotinamide adenine dinucleotide and poly(adenosine diphosphoribose) metabolism by the synthetic "C" nucleoside analogs, tiazofurin and selenazofurin. A new strategy for cancer chemotherapy. J Clin Invest, 75(2), 702-709. doi: 10.1172/JCI111750 Bjorkoy, G., Lamark, T., Brech, A., Outzen, H., Perander, M., Overvatn, A., . . . Johansen, T. (2005). p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol, 171(4), 603-614. doi: 10.1083/jcb.200507002 Bjorkoy, G., Lamark, T., Pankiv, S., Overvatn, A., Brech, A., & Johansen, T. (2009). Monitoring autophagic degradation of p62/SQSTM1. Methods Enzymol, 452, 181-197. doi: 10.1016/S0076-6879(08)03612-4 Blommaart, E. F., Luiken, J. J., Blommaart, P. J., van Woerkom, G. M., & Meijer, A. J. (1995). Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. J Biol Chem, 270(5), 2320- 2326. Borgers, M., & De Nollin, S. (1975). Ultrastructural changes in Ascaris suum intestine after mebendazole treatment in vivo. J Parasitol, 61(1), 110- 122. Bowe, D. B., Kenney, N. J., Adereth, Y., & Maroulakou, I. G. (2002). Suppression of Neu-induced mammary tumor growth in cyclin D1 deficient mice is compensated for by cyclin E. Oncogene, 21(2), 291- 298. doi: 10.1038/sj.onc.1205025 Bracho-Valdes, I., Moreno-Alvarez, P., Valencia-Martinez, I., Robles-Molina, E., Chavez-Vargas, L., & Vazquez-Prado, J. (2011). mTORC1- and mTORC2-interacting proteins keep their multifunctional partners focused. IUBMB Life, 63(10), 896-914. doi: 10.1002/iub.558 Page 184 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Brustmann, H. (2007). Poly(adenosine diphosphate-ribose) polymerase expression in serous ovarian carcinoma: correlation with p53, MIB-1, and outcome. Int J Gynecol Pathol, 26(2), 147-153. doi: 10.1097/01.pgp.0000235064.93182.ec Buys, S. S., Partridge, E., Black, A., Johnson, C. C., Lamerato, L., Isaacs, C., . . . Team, P. P. (2011). Effect of screening on ovarian cancer mortality: the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Randomized Controlled Trial. JAMA, 305(22), 2295-2303. doi: 10.1001/jama.2011.766 Cantley, L. C. (2002). The phosphoinositide 3-kinase pathway. SCIENCE, 296(5573), 1655-1657. doi: 10.1126/science.296.5573.1655 Castino, R., Bellio, N., Follo, C., Murphy, D., & Isidoro, C. (2010). Inhibition of PI3k class III-dependent autophagy prevents apoptosis and necrosis by oxidative stress in dopaminergic neuroblastoma cells. Toxicol Sci, 117(1), 152-162. doi: 10.1093/toxsci/kfq170 Cecchini, M. J., Amiri, M., & Dick, F. A. (2012). Analysis of cell cycle position in mammalian cells. J Vis Exp(59). doi: 10.3791/3491 Chaabane, W., User, S. D., El-Gazzah, M., Jaksik, R., Sajjadi, E., Rzeszowska- Wolny, J., & Los, M. J. (2013). Autophagy, apoptosis, mitoptosis and necrosis: interdependence between those pathways and effects on cancer. Arch Immunol Ther Exp (Warsz), 61(1), 43-58. doi: 10.1007/s00005-012-0205-y Chaitanya, G. V., Steven, A. J., & Babu, P. P. (2010). PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration. Cell Commun Signal, 8, 31. doi: 10.1186/1478-811X-8-31 Chen, H. Y., & White, E. (2011). Role of autophagy in cancer prevention. Cancer Prev Res (Phila), 4(7), 973-983. doi: 10.1158/1940-6207.CAPR- 10-0387 Chen, N., & Karantza-Wadsworth, V. (2009). Role and regulation of autophagy in cancer. Biochim Biophys Acta, 1793(9), 1516-1523. doi: 10.1016/j.bbamcr.2008.12.013 Page 185 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Chresta, C. M., Davies, B. R., Hickson, I., Harding, T., Cosulich, S., Critchlow, S. E., . . . Pass, M. (2010). AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity. Cancer Res, 70(1), 288-298. doi: 10.1158/0008-5472.CAN-09-1751 Clarke-Pearson, D. L. (2009). Clinical practice. Screening for ovarian cancer. N Engl J Med, 361(2), 170-177. doi: 10.1056/NEJMcp0901926 Cloughesy, T. F., Yoshimoto, K., Nghiemphu, P., Brown, K., Dang, J., Zhu, S., . . . Sawyers, C. L. (2008). Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma. PLoS Med, 5(1), e8. doi: 10.1371/journal.pmed.0050008 Colombi, M., Molle, K. D., Benjamin, D., Rattenbacher-Kiser, K., Schaefer, C., Betz, C., . . . Moroni, C. (2011). Genome-wide shRNA screen reveals increased mitochondrial dependence upon mTORC2 addiction. Oncogene, 30(13), 1551-1565. doi: 10.1038/onc.2010.539 Cramer, D. W., & Welch, W. R. (1983). Determinants of ovarian cancer risk. II. Inferences regarding pathogenesis. J Natl Cancer Inst, 71(4), 717-721. Dadak, A. M., Asanger, H., Tichy, A., & Franz, S. (2013). Establishing an efficacious dose rate of monepantel for treating gastrointestinal nematodes in llamas under field conditions. Vet Rec, 172(6), 155. doi: 10.1136/vr.101109 De Benedetti, A., & Graff, J. R. (2004). eIF-4E expression and its role in malignancies and metastases. Oncogene, 23(18), 3189-3199. doi: 10.1038/sj.onc.1207545 de Fries, R., & Mitsuhashi, M. (1995). Quantification of mitogen induced human lymphocyte proliferation: comparison of alamarBlue assay to 3H- thymidine incorporation assay. J Clin Lab Anal, 9(2), 89-95. Degenhardt, K., & White, E. (2006). A mouse model system to genetically dissect the molecular mechanisms regulating tumorigenesis. Clin Cancer Res, 12(18), 5298-5304. doi: 10.1158/1078-0432.CCR-06-0439 Page 186 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Dong, J., Peng, J., Zhang, H., Mondesire, W. H., Jian, W., Mills, G. B., . . . Meric-Bernstam, F. (2005). Role of glycogen synthase kinase 3beta in rapamycin-mediated cell cycle regulation and chemosensitivity. Cancer Res, 65(5), 1961-1972. doi: 10.1158/0008-5472.CAN-04-2501 Donnelly-Roberts, D. L., & Lentz, T. L. (1991). Binding sites for alpha- bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the alpha-subunit of the nicotinic acetylcholine receptor. Biochemistry, 30(30), 7484-7491. Ducray, P., Gauvry, N., Pautrat, F., Goebel, T., Fruechtel, J., Desaules, Y., . . . Kaminsky, R. (2008). Discovery of amino-acetonitrile derivatives, a new class of synthetic anthelmintic compounds. Bioorg Med Chem Lett, 18(9), 2935-2938. doi: 10.1016/j.bmcl.2008.03.071 Dufner, A., & Thomas, G. (1999). Ribosomal S6 kinase signaling and the control of translation. Exp Cell Res, 253(1), 100-109. doi: 10.1006/excr.1999.4683 Duran, A., Linares, J. F., Galvez, A. S., Wikenheiser, K., Flores, J. M., Diaz- Meco, M. T., & Moscat, J. (2008). The signaling adaptor p62 is an important NF-kappaB mediator in tumorigenesis. Cancer Cell, 13(4), 343-354. doi: 10.1016/j.ccr.2008.02.001 Duronio, V. (2008). The life of a cell: apoptosis regulation by the PI3K/PKB pathway. Biochem J, 415(3), 333-344. doi: 10.1042/BJ20081056 Duvel, K., Yecies, J. L., Menon, S., Raman, P., Lipovsky, A. I., Souza, A. L., . . . Manning, B. D. (2010). Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol Cell, 39(2), 171-183. doi: 10.1016/j.molcel.2010.06.022 Efeyan, A., & Sabatini, D. M. (2010). mTOR and cancer: many loops in one pathway. Curr Opin Cell Biol, 22(2), 169-176. doi: 10.1016/j.ceb.2009.10.007 Egan, D. F., Shackelford, D. B., Mihaylova, M. M., Gelino, S., Kohnz, R. A., Mair, W., . . . Shaw, R. J. (2011). Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. SCIENCE, 331(6016), 456-461. doi: 10.1126/science.1196371 Page 187 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Espey, L. L. (1994). Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction. Biol Reprod, 50(2), 233-238. Estaquier, J., Vallette, F., Vayssiere, J. L., & Mignotte, B. (2012). The mitochondrial pathways of apoptosis. Adv Exp Med Biol, 942, 157-183. doi: 10.1007/978-94-007-2869-1_7 Fathalla, M. F. (1971). Incessant ovulation--a factor in ovarian neoplasia? Lancet, 2(7716), 163. Feldman, M. E., Apsel, B., Uotila, A., Loewith, R., Knight, Z. A., Ruggero, D., & Shokat, K. M. (2009). Active-site inhibitors of mTOR target rapamycin- resistant outputs of mTORC1 and mTORC2. PLoS Biol, 7(2), e38. doi: 10.1371/journal.pbio.1000038 Foster, D. A., Yellen, P., Xu, L., & Saqcena, M. (2010). Regulation of G1 Cell Cycle Progression: Distinguishing the Restriction Point from a Nutrient- Sensing Cell Growth Checkpoint(s). Genes Cancer, 1(11), 1124-1131. doi: 10.1177/1947601910392989 Frias, M. A., Thoreen, C. C., Jaffe, J. D., Schroder, W., Sculley, T., Carr, S. A., & Sabatini, D. M. (2006). mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s. Curr Biol, 16(18), 1865-1870. doi: 10.1016/j.cub.2006.08.001 Fruman, D. A., & Rommel, C. (2014). PI3K and cancer: lessons, challenges and opportunities. Nat Rev Drug Discov, 13(2), 140-156. doi: 10.1038/nrd4204 Fujita, N., Hayashi-Nishino, M., Fukumoto, H., Omori, H., Yamamoto, A., Noda, T., & Yoshimori, T. (2008). An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure. Mol Biol Cell, 19(11), 4651-4659. doi: 10.1091/mbc.E08-03-0312 Fumarola, C., Bonelli, M. A., Petronini, P. G., & Alfieri, R. R. (2014). Targeting PI3K/AKT/mTOR pathway in non small cell lung cancer. Biochem Pharmacol, 90(3), 197-207. doi: 10.1016/j.bcp.2014.05.011 Page 188 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Gan, X., Wang, J., Su, B., & Wu, D. (2011). Evidence for direct activation of mTORC2 kinase activity by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem, 286(13), 10998-11002. doi: 10.1074/jbc.M110.195016 Garcia-Martinez, J. M., & Alessi, D. R. (2008). mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1). Biochem J, 416(3), 375- 385. doi: 10.1042/BJ20081668 Goncharova, E. A., Goncharov, D. A., Li, H., Pimtong, W., Lu, S., Khavin, I., & Krymskaya, V. P. (2011). mTORC2 is required for proliferation and survival of TSC2-null cells. Mol Cell Biol, 31(12), 2484-2498. doi: 10.1128/MCB.01061-10 Gu, Y., Lindner, J., Kumar, A., Yuan, W., & Magnuson, M. A. (2011). Rictor/mTORC2 is essential for maintaining a balance between beta-cell proliferation and cell size. Diabetes, 60(3), 827-837. doi: 10.2337/db10- 1194 Gubbels, J. A., Claussen, N., Kapur, A. K., Connor, J. P., & Patankar, M. S. (2010). The detection, treatment, and biology of epithelial ovarian cancer. J Ovarian Res, 3, 8. doi: 10.1186/1757-2215-3-8 Guertin, D. A., & Sabatini, D. M. (2009). The pharmacology of mTOR inhibition. Sci Signal, 2(67), pe24. doi: 10.1126/scisignal.267pe24 Guertin, D. A., Stevens, D. M., Thoreen, C. C., Burds, A. A., Kalaany, N. Y., Moffat, J., . . . Sabatini, D. M. (2006). Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. Dev Cell, 11(6), 859-871. doi: 10.1016/j.devcel.2006.10.007 Gulhati, P., Bowen, K. A., Liu, J., Stevens, P. D., Rychahou, P. G., Chen, M., . . . Evers, B. M. (2011). mTORC1 and mTORC2 regulate EMT, motility, and metastasis of colorectal cancer via RhoA and Rac1 signaling pathways. Cancer Res, 71(9), 3246-3256. doi: 10.1158/0008-5472.CAN- 10-4058 Guo, J. Y., Chen, H. Y., Mathew, R., Fan, J., Strohecker, A. M., Karsli-Uzunbas, G., . . . White, E. (2011). Activated Ras requires autophagy to maintain Page 189 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer oxidative metabolism and tumorigenesis. Genes Dev, 25(5), 460-470. doi: 10.1101/gad.2016311 Hagiwara, A., Cornu, M., Cybulski, N., Polak, P., Betz, C., Trapani, F., . . . Hall, M. N. (2012). Hepatic mTORC2 activates glycolysis and lipogenesis through Akt, glucokinase, and SREBP1c. Cell Metab, 15(5), 725-738. doi: 10.1016/j.cmet.2012.03.015 Halevi, S., McKay, J., Palfreyman, M., Yassin, L., Eshel, M., Jorgensen, E., & Treinin, M. (2002). The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors. EMBO J, 21(5), 1012-1020. doi: 10.1093/emboj/21.5.1012 Han, E. S., Wakabayashi, M., & Leong, L. (2013). Angiogenesis inhibitors in the treatment of epithelial ovarian cancer. Curr Treat Options Oncol, 14(1), 22-33. doi: 10.1007/s11864-012-0220-6 Hara, T., Nakamura, K., Matsui, M., Yamamoto, A., Nakahara, Y., Suzuki- Migishima, R., . . . Mizushima, N. (2006). Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature, 441(7095), 885-889. doi: 10.1038/nature04724 Harrison, D. E., Strong, R., Sharp, Z. D., Nelson, J. F., Astle, C. M., Flurkey, K., . . . Miller, R. A. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. nature, 460(7253), 392-395. doi: 10.1038/nature08221 Hay, N., & Sonenberg, N. (2004). Upstream and downstream of mTOR. Genes Dev, 18(16), 1926-1945. doi: 10.1101/gad.1212704 Hayun, R., Okun, E., Berrebi, A., Shvidel, L., Bassous, L., Sredni, B., & Nir, U. (2009). Rapamycin and curcumin induce apoptosis in primary resting B chronic lymphocytic leukemia cells. Leuk Lymphoma, 50(4), 625-632. doi: 10.1080/10428190902789181 Hennessy, B. T., Coleman, R. L., & Markman, M. (2009). Ovarian cancer. Lancet, 374(9698), 1371-1382. doi: 10.1016/S0140-6736(09)61338-6 Hershey, J. W. (2010). Regulation of protein synthesis and the role of eIF3 in cancer. Braz J Med Biol Res, 43(10), 920-930. Page 190 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Hosking, B. C., Griffiths, T. M., Woodgate, R. G., Besier, R. B., Le Feuvre, A. S., Nilon, P., . . . Seewald, W. (2009). Clinical field study to evaluate the efficacy and safety of the amino-acetonitrile derivative, monepantel, compared with registered anthelmintics against gastrointestinal nematodes of sheep in Australia. Aust Vet J, 87(11), 455-462. doi: 10.1111/j.1751-0813.2009.00511.x Hosking, B. C., Kaminsky, R., Sager, H., Rolfe, P. F., & Seewald, W. (2010). A pooled analysis of the efficacy of monepantel, an amino-acetonitrile derivative against gastrointestinal nematodes of sheep. Parasitol Res, 106(2), 529-532. doi: 10.1007/s00436-009-1636-1 Hosokawa, N., Hara, T., Kaizuka, T., Kishi, C., Takamura, A., Miura, Y., . . . Mizushima, N. (2009). Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell, 20(7), 1981-1991. doi: 10.1091/mbc.E08-12-1248 Houghton, P. J. (2010). Everolimus. Clin Cancer Res, 16(5), 1368-1372. doi: 10.1158/1078-0432.CCR-09-1314 Hsieh, A. C., Liu, Y., Edlind, M. P., Ingolia, N. T., Janes, M. R., Sher, A., . . . Ruggero, D. (2012). The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature, 485(7396), 55-61. doi: 10.1038/nature10912 Hu, L., Hofmann, J., Lu, Y., Mills, G. B., & Jaffe, R. B. (2002). Inhibition of phosphatidylinositol 3'-kinase increases efficacy of paclitaxel in in vitro and in vivo ovarian cancer models. Cancer Res, 62(4), 1087-1092. Huang, J., Dibble, C. C., Matsuzaki, M., & Manning, B. D. (2008). The TSC1- TSC2 complex is required for proper activation of mTOR complex 2. Mol Cell Biol, 28(12), 4104-4115. doi: 10.1128/MCB.00289-08 Ichimura, Y., Kominami, E., Tanaka, K., & Komatsu, M. (2008). Selective turnover of p62/A170/SQSTM1 by autophagy. Autophagy, 4(8), 1063- 1066. Ichimura, Y., Kumanomidou, T., Sou, Y. S., Mizushima, T., Ezaki, J., Ueno, T., . . . Komatsu, M. (2008). Structural basis for sorting mechanism of p62 in selective autophagy. J Biol Chem, 283(33), 22847-22857. doi: 10.1074/jbc.M802182200 Page 191 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Ikenoue, T., Inoki, K., Yang, Q., Zhou, X., & Guan, K. L. (2008). Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling. EMBO J, 27(14), 1919-1931. doi: 10.1038/emboj.2008.119 Inoki, K., Corradetti, M. N., & Guan, K. L. (2005). Dysregulation of the TSC- mTOR pathway in human disease. Nat Genet, 37(1), 19-24. doi: 10.1038/ng1494 Inoki, K., Zhu, T., & Guan, K. L. (2003). TSC2 mediates cellular energy response to control cell growth and survival. Cell, 115(5), 577-590. Jacinto, E., Facchinetti, V., Liu, D., Soto, N., Wei, S., Jung, S. Y., . . . Su, B. (2006). SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell, 127(1), 125- 137. doi: 10.1016/j.cell.2006.08.033 Jacinto, E., Loewith, R., Schmidt, A., Lin, S., Ruegg, M. A., Hall, A., & Hall, M. N. (2004). Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol, 6(11), 1122-1128. doi: 10.1038/ncb1183 Jain, A., Lamark, T., Sjottem, E., Larsen, K. B., Awuh, J. A., Overvatn, A., . . . Johansen, T. (2010). p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription. J Biol Chem, 285(29), 22576-22591. doi: 10.1074/jbc.M110.118976 Jeong, J. B., Hong, S. C., Jeong, H. J., & Koo, J. S. (2011). Arctigenin induces cell cycle arrest by blocking the phosphorylation of Rb via the modulation of cell cycle regulatory proteins in human gastric cancer cells. Int Immunopharmacol, 11(10), 1573-1577. doi: 10.1016/j.intimp.2011.05.016 Jones, M. D., Hunter, R. P., Dobson, D. P., Reymond, N., Strehlau, G. A., Kubacki, P., . . . Walters, M. E. (2010). European field study of the efficacy and safety of the novel anthelmintic monepantel in sheep. Vet Rec, 167(16), 610-613. doi: 10.1136/vr.c4477 Jung, C. H., Jun, C. B., Ro, S. H., Kim, Y. M., Otto, N. M., Cao, J., . . . Kim, D. H. (2009). ULK-Atg13-FIP200 complexes mediate mTOR signaling to the Page 192 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer autophagy machinery. Mol Biol Cell, 20(7), 1992-2003. doi: 10.1091/mbc.E08-12-1249 Kadowaki, M., & Karim, M. R. (2009). Cytosolic LC3 ratio as a quantitative index of macroautophagy. Methods Enzymol, 452, 199-213. doi: 10.1016/S0076-6879(08)03613-6 Kamada, Y., Yoshino, K., Kondo, C., Kawamata, T., Oshiro, N., Yonezawa, K., & Ohsumi, Y. (2010). Tor directly controls the Atg1 kinase complex to regulate autophagy. Mol Cell Biol, 30(4), 1049-1058. doi: 10.1128/MCB.01344-09 Kaminsky, R., Bapst, B., Stein, P. A., Strehlau, G. A., Allan, B. A., Hosking, B. C., . . . Sager, H. (2011). Differences in efficacy of monepantel, derquantel and abamectin against multi-resistant nematodes of sheep. Parasitol Res, 109(1), 19-23. doi: 10.1007/s00436-010-2216-0 Kaminsky, R., Ducray, P., Jung, M., Clover, R., Rufener, L., Bouvier, J., . . . Maser, P. (2008). A new class of anthelmintics effective against drug- resistant nematodes. Nature, 452(7184), 176-180. doi: 10.1038/nature06722 Kaminsky, R., Mosimann, D., Sager, H., Stein, P., & Hosking, B. (2009). Determination of the effective dose rate for monepantel (AAD 1566) against adult gastro-intestinal nematodes in sheep. Int J Parasitol, 39(4), 443-446. doi: 10.1016/j.ijpara.2008.09.009 Kapoor, A. (2009). Inhibition of mTOR in kidney cancer. Curr Oncol, 16 Suppl 1, S33-39. Karadzovska, D., Seewald, W., Browning, A., Smal, M., Bouvier, J., & Giraudel, J. M. (2009). Pharmacokinetics of monepantel and its sulfone metabolite, monepantel sulfone, after intravenous and oral administration in sheep. J Vet Pharmacol Ther, 32(4), 359-367. doi: 10.1111/j.1365- 2885.2008.01052.x Katso, R., Okkenhaug, K., Ahmadi, K., White, S., Timms, J., & Waterfield, M. D. (2001). Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol, 17, 615-675. doi: 10.1146/annurev.cellbio.17.1.615 Page 193 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Khan, A. A., Abel, P. D., Chaudhary, K. S., Gulzar, Z., Stamp, G. W., & Lalani, E. N. (2003). Inverse correlation between high level expression of cyclin E and proliferation index in transitional cell carcinoma of the bladder. Mol Pathol, 56(6), 353-361. Kim, J., Kundu, M., Viollet, B., & Guan, K. L. (2011). AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol, 13(2), 132-141. doi: 10.1038/ncb2152 King, J. S. (2012). Mechanical stress meets autophagy: potential implications for physiology and pathology. Trends Mol Med, 18(10), 583-588. doi: 10.1016/j.molmed.2012.08.002 Kirisako, T., Ichimura, Y., Okada, H., Kabeya, Y., Mizushima, N., Yoshimori, T., . . . Ohsumi, Y. (2000). The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. J Cell Biol, 151(2), 263-276. Kirkin, V., Lamark, T., Johansen, T., & Dikic, I. (2009). NBR1 cooperates with p62 in selective autophagy of ubiquitinated targets. Autophagy, 5(5), 732-733. Kisielewski, R., Tolwinska, A., Mazurek, A., & Laudanski, P. (2013). Inflammation and ovarian cancer--current views. Ginekol Pol, 84(4), 293- 297. Komatsu, M., Waguri, S., Koike, M., Sou, Y. S., Ueno, T., Hara, T., . . . Tanaka, K. (2007). Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell, 131(6), 1149-1163. doi: 10.1016/j.cell.2007.10.035 Kroemer, G., Galluzzi, L., Vandenabeele, P., Abrams, J., Alnemri, E. S., Baehrecke, E. H., . . . Nomenclature Committee on Cell, D. (2009). Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ, 16(1), 3-11. doi: 10.1038/cdd.2008.150 Kuma, A., Hatano, M., Matsui, M., Yamamoto, A., Nakaya, H., Yoshimori, T., . . . Mizushima, N. (2004). The role of autophagy during the early neonatal starvation period. Nature, 432(7020), 1032-1036. doi: 10.1038/nature03029 Page 194 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Kuma, A., & Mizushima, N. (2010). Physiological role of autophagy as an intracellular recycling system: with an emphasis on nutrient metabolism. Semin Cell Dev Biol, 21(7), 683-690. doi: 10.1016/j.semcdb.2010.03.002 Lai, D., Visser-Grieve, S., & Yang, X. (2012). Tumour suppressor genes in chemotherapeutic drug response. Biosci Rep, 32(4), 361-374. doi: 10.1042/BSR20110125 Lang, F., Bohmer, C., Palmada, M., Seebohm, G., Strutz-Seebohm, N., & Vallon, V. (2006). (Patho)physiological significance of the serum- and glucocorticoid-inducible kinase isoforms. Physiol Rev, 86(4), 1151-1178. doi: 10.1152/physrev.00050.2005 Laplante, M., & Sabatini, D. M. (2009). An emerging role of mTOR in lipid biosynthesis. Curr Biol, 19(22), R1046-1052. doi: 10.1016/j.cub.2009.09.058 Laplante, M., & Sabatini, D. M. (2012). mTOR signaling in growth control and disease. Cell, 149(2), 274-293. doi: 10.1016/j.cell.2012.03.017 Larsson, O., Perlman, D. M., Fan, D., Reilly, C. S., Peterson, M., Dahlgren, C., . . . Bitterman, P. B. (2006). Apoptosis resistance downstream of eIF4E: posttranscriptional activation of an anti-apoptotic transcript carrying a consensus hairpin structure. Nucleic Acids Res, 34(16), 4375-4386. doi: 10.1093/nar/gkl558 Lau, A., Wang, X. J., Zhao, F., Villeneuve, N. F., Wu, T., Jiang, T., . . . Zhang, D. D. (2010). A noncanonical mechanism of Nrf2 activation by autophagy deficiency: direct interaction between Keap1 and p62. Mol Cell Biol, 30(13), 3275-3285. doi: 10.1128/MCB.00248-10 Lee, Y., Miron, A., Drapkin, R., Nucci, M. R., Medeiros, F., Saleemuddin, A., . . . Crum, C. P. (2007). A candidate precursor to serous carcinoma that originates in the distal fallopian tube. J Pathol, 211(1), 26-35. doi: 10.1002/path.2091 Levine, B., & Kroemer, G. (2008). Autophagy in the pathogenesis of disease. Cell, 132(1), 27-42. doi: 10.1016/j.cell.2007.12.018 Page 195 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Li, D., Liu, X., Lin, L., Hou, J., Li, N., Wang, C., . . . Cao, X. (2011). MicroRNA- 99a inhibits hepatocellular carcinoma growth and correlates with prognosis of patients with hepatocellular carcinoma. J Biol Chem, 286(42), 36677-36685. doi: 10.1074/jbc.M111.270561 Lifschitz, A., Ballent, M., Virkel, G., Sallovitz, J., Viviani, P., & Lanusse, C. (2014). Accumulation of monepantel and its sulphone derivative in tissues of nematode location in sheep: pharmacokinetic support to its excellent nematodicidal activity. Vet Parasitol, 203(1-2), 120-126. doi: 10.1016/j.vetpar.2014.02.049 Lippai, M., & Low, P. (2014). The role of the selective adaptor p62 and ubiquitin- like proteins in autophagy. Biomed Res Int, 2014, 832704. doi: 10.1155/2014/832704 Liu, L., Das, S., Losert, W., & Parent, C. A. (2010). mTORC2 regulates neutrophil chemotaxis in a cAMP- and RhoA-dependent fashion. Dev Cell, 19(6), 845-857. doi: 10.1016/j.devcel.2010.11.004 Liu, Q., Thoreen, C., Wang, J., Sabatini, D., & Gray, N. S. (2009). mTOR Mediated Anti-Cancer Drug Discovery. Drug Discov Today Ther Strateg, 6(2), 47-55. doi: 10.1016/j.ddstr.2009.12.001 Lu, M., Wang, J., Jones, K. T., Ives, H. E., Feldman, M. E., Yao, L. J., . . . Pearce, D. (2010). mTOR complex-2 activates ENaC by phosphorylating SGK1. J Am Soc Nephrol, 21(5), 811-818. doi: 10.1681/ASN.2009111168 Lum, J. J., Bauer, D. E., Kong, M., Harris, M. H., Li, C., Lindsten, T., & Thompson, C. B. (2005). Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell, 120(2), 237-248. doi: 10.1016/j.cell.2004.11.046 Ma, X. M., & Blenis, J. (2009). Molecular mechanisms of mTOR-mediated translational control. Nat Rev Mol Cell Biol, 10(5), 307-318. doi: 10.1038/nrm2672 Maclean, K. H., Dorsey, F. C., Cleveland, J. L., & Kastan, M. B. (2008). Targeting lysosomal degradation induces p53-dependent cell death and prevents cancer in mouse models of lymphomagenesis. J Clin Invest, 118(1), 79-88. doi: 10.1172/JCI33700 Page 196 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Maes, H., Rubio, N., Garg, A. D., & Agostinis, P. (2013). Autophagy: shaping the tumor microenvironment and therapeutic response. Trends Mol Med, 19(7), 428-446. doi: 10.1016/j.molmed.2013.04.005 Maiuri, M. C., Zalckvar, E., Kimchi, A., & Kroemer, G. (2007). Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol, 8(9), 741-752. doi: 10.1038/nrm2239 Majmundar, A. J., Wong, W. J., & Simon, M. C. (2010). Hypoxia-inducible factors and the response to hypoxic stress. Mol Cell, 40(2), 294-309. doi: 10.1016/j.molcel.2010.09.022 Malikides, N., Helbig, R., Roth, D. R., Alexander, A., Hosking, B. C., & Strehlau, G. A. (2009). Safety of an amino-acetonitrile derivative ( AAD ), monepantel, in weaned lambs following repeated oral administration. N Z Vet J, 57(1), 10-15. doi: 10.1080/00480169.2009.36862 Malumbres, M., & Barbacid, M. (2009). Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer, 9(3), 153-166. doi: 10.1038/nrc2602 Mamane, Y., Petroulakis, E., Martineau, Y., Sato, T. A., Larsson, O., Rajasekhar, V. K., & Sonenberg, N. (2007). Epigenetic activation of a subset of mRNAs by eIF4E explains its effects on cell proliferation. PLoS One, 2(2), e242. doi: 10.1371/journal.pone.0000242 Mammucari, C., Milan, G., Romanello, V., Masiero, E., Rudolf, R., Del Piccolo, P., . . . Sandri, M. (2007). FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab, 6(6), 458-471. doi: 10.1016/j.cmet.2007.11.001 Manning, B. D. (2004). Balancing Akt with S6K: implications for both metabolic diseases and tumorigenesis. J Cell Biol, 167(3), 399-403. doi: 10.1083/jcb.200408161 Martin, D. E., & Hall, M. N. (2005). The expanding TOR signaling network. Curr Opin Cell Biol, 17(2), 158-166. doi: 10.1016/j.ceb.2005.02.008 Martin, R. J. (1997). Modes of action of anthelmintic drugs. Vet J, 154(1), 11-34. Page 197 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Masamha, C. P., & Benbrook, D. M. (2009). Cyclin D1 degradation is sufficient to induce G1 cell cycle arrest despite constitutive expression of cyclin E2 in ovarian cancer cells. Cancer Res, 69(16), 6565-6572. doi: 10.1158/0008-5472.CAN-09-0913 Mathew, R., Karp, C. M., Beaudoin, B., Vuong, N., Chen, G., Chen, H. Y., . . . White, E. (2009). Autophagy suppresses tumorigenesis through elimination of p62. Cell, 137(6), 1062-1075. doi: 10.1016/j.cell.2009.03.048 Mathew, R., Kongara, S., Beaudoin, B., Karp, C. M., Bray, K., Degenhardt, K., . . . White, E. (2007). Autophagy suppresses tumor progression by limiting chromosomal instability. Genes Dev, 21(11), 1367-1381. doi: 10.1101/gad.1545107 Matsunaga, K., Saitoh, T., Tabata, K., Omori, H., Satoh, T., Kurotori, N., . . . Yoshimori, T. (2009). Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nat Cell Biol, 11(4), 385-396. doi: 10.1038/ncb1846 Matsushime, H., Quelle, D. E., Shurtleff, S. A., Shibuya, M., Sherr, C. J., & Kato, J. Y. (1994). D-type cyclin-dependent kinase activity in mammalian cells. Mol Cell Biol, 14(3), 2066-2076. Mayer, C., Zhao, J., Yuan, X., & Grummt, I. (2004). mTOR-dependent activation of the transcription factor TIF-IA links rRNA synthesis to nutrient availability. Genes Dev, 18(4), 423-434. doi: 10.1101/gad.285504 Mazure, N. M., & Pouyssegur, J. (2010). Hypoxia-induced autophagy: cell death or cell survival? Curr Opin Cell Biol, 22(2), 177-180. doi: 10.1016/j.ceb.2009.11.015 McIlwain, D. R., Berger, T., & Mak, T. W. (2013). Caspase functions in cell death and disease. Cold Spring Harb Perspect Biol, 5(4), a008656. doi: 10.1101/cshperspect.a008656 Mercer, C. A., Kaliappan, A., & Dennis, P. B. (2009). A novel, human Atg13 binding protein, Atg101, interacts with ULK1 and is essential for macroautophagy. Autophagy, 5(5), 649-662. Page 198 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Meric-Bernstam, F., & Gonzalez-Angulo, A. M. (2009). Targeting the mTOR signaling network for cancer therapy. J Clin Oncol, 27(13), 2278-2287. doi: 10.1200/JCO.2008.20.0766 Mitchell, C. L., O'Connor, J. P., Jackson, A., Parker, G. J., Roberts, C., Watson, Y., . . . Jayson, G. C. (2010). Identification of early predictive imaging biomarkers and their relationship to serological angiogenic markers in patients with ovarian cancer with residual disease following cytotoxic therapy. Ann Oncol, 21(10), 1982-1989. doi: 10.1093/annonc/mdq079 Mizushima, N., & Levine, B. (2010). Autophagy in mammalian development and differentiation. Nat Cell Biol, 12(9), 823-830. doi: 10.1038/ncb0910-823 Mizushima, N., & Yoshimori, T. (2007). How to interpret LC3 immunoblotting. Autophagy, 3(6), 542-545. Mizushima, N., Yoshimori, T., & Levine, B. (2010). Methods in mammalian autophagy research. Cell, 140(3), 313-326. doi: 10.1016/j.cell.2010.01.028 Mongan, N. P., Jones, A. K., Smith, G. R., Sansom, M. S., & Sattelle, D. B. (2002). Novel alpha7-like nicotinic acetylcholine receptor subunits in the nematode Caenorhabditis elegans. Protein Sci, 11(5), 1162-1171. doi: 10.1110/ps.3040102 Montero, J., Dutta, C., van Bodegom, D., Weinstock, D., & Letai, A. (2013). p53 regulates a non-apoptotic death induced by ROS. Cell Death Differ, 20(11), 1465-1474. doi: 10.1038/cdd.2013.52 Moretti, L., Yang, E. S., Kim, K. W., & Lu, B. (2007). Autophagy signaling in cancer and its potential as novel target to improve anticancer therapy. Drug Resist Updat, 10(4-5), 135-143. doi: 10.1016/j.drup.2007.05.001 Morgan, T. M., Koreckij, T. D., & Corey, E. (2009). Targeted therapy for advanced prostate cancer: inhibition of the PI3K/Akt/mTOR pathway. Curr Cancer Drug Targets, 9(2), 237-249. Moscat, J., Diaz-Meco, M. T., & Wooten, M. W. (2007). Signal integration and diversification through the p62 scaffold protein. Trends Biochem Sci, 32(2), 95-100. doi: 10.1016/j.tibs.2006.12.002 Page 199 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Mukhopadhyay, N. K., Price, D. J., Kyriakis, J. M., Pelech, S., Sanghera, J., & Avruch, J. (1992). An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase. J Biol Chem, 267(5), 3325-3335. Naito, S., Koike, K., Ono, M., Machida, T., Tasaka, S., Kiue, A., . . . Kumazawa, J. (1998). Development of novel reversal agents, imidazothiazole derivatives, targeting MDR1- and MRP-mediated multidrug resistance. Oncol Res, 10(3), 123-132. Nakayama, K. I., & Nakayama, K. (2006). Ubiquitin ligases: cell-cycle control and cancer. Nat Rev Cancer, 6(5), 369-381. doi: 10.1038/nrc1881 Narasimha, A. M., Kaulich, M., Shapiro, G. S., Choi, Y. J., Sicinski, P., & Dowdy, S. F. (2014). Cyclin D activates the Rb tumor suppressor by mono-phosphorylation. Elife, 3. doi: 10.7554/eLife.02872 Neufeld, T. P. (2010). TOR-dependent control of autophagy: biting the hand that feeds. Curr Opin Cell Biol, 22(2), 157-168. doi: 10.1016/j.ceb.2009.11.005 Neufeld, T. P. (2012). Autophagy and cell growth--the yin and yang of nutrient responses. J Cell Sci, 125(Pt 10), 2359-2368. doi: 10.1242/jcs.103333 Nezhat, F., Datta, M. S., Hanson, V., Pejovic, T., Nezhat, C., & Nezhat, C. (2008). The relationship of endometriosis and ovarian malignancy: a review. Fertil Steril, 90(5), 1559-1570. doi: 10.1016/j.fertnstert.2008.08.007 Nixon, R. A. (2013). The role of autophagy in neurodegenerative disease. Nat Med, 19(8), 983-997. doi: 10.1038/nm.3232 Noguchi, Y., Young, J. D., Aleman, J. O., Hansen, M. E., Kelleher, J. K., & Stephanopoulos, G. (2009). Effect of anaplerotic fluxes and amino acid availability on hepatic lipoapoptosis. J Biol Chem, 284(48), 33425-33436. doi: 10.1074/jbc.M109.049478 O'Reilly, K. E., Rojo, F., She, Q. B., Solit, D., Mills, G. B., Smith, D., . . . Rosen, N. (2006). mTOR inhibition induces upstream receptor tyrosine kinase Page 200 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer signaling and activates Akt. Cancer Res, 66(3), 1500-1508. doi: 10.1158/0008-5472.CAN-05-2925 Oh, W. J., & Jacinto, E. (2011). mTOR complex 2 signaling and functions. Cell Cycle, 10(14), 2305-2316. Oh, W. J., Wu, C. C., Kim, S. J., Facchinetti, V., Julien, L. A., Finlan, M., . . . Jacinto, E. (2010). mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide. EMBO J, 29(23), 3939-3951. doi: 10.1038/emboj.2010.271 Ohsumi, Y. (2014). Historical landmarks of autophagy research. Cell Res, 24(1), 9-23. doi: 10.1038/cr.2013.169 Oku, M., & Sakai, Y. (2010). Peroxisomes as dynamic organelles: autophagic degradation. FEBS J, 277(16), 3289-3294. doi: 10.1111/j.1742- 4658.2010.07741.x Oliver, F. J., de la Rubia, G., Rolli, V., Ruiz-Ruiz, M. C., de Murcia, G., & Murcia, J. M. (1998). Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant. J Biol Chem, 273(50), 33533-33539. Orvieto, R., Fisch, N., Yulzari-Roll, V., & La Marca, A. (2005). Ovarian androgens but not estrogens correlate with the degree of systemic inflammation observed during controlled ovarian hyperstimulation. Gynecol Endocrinol, 21(3), 170-173. doi: 10.1080/09513590500279667 Pacheco, C. D., & Lieberman, A. P. (2008). The pathogenesis of Niemann-Pick type C disease: a role for autophagy? Expert Rev Mol Med, 10, e26. doi: 10.1017/S146239940800080X Pagano, A., Metrailler-Ruchonnet, I., Aurrand-Lions, M., Lucattelli, M., Donati, Y., & Argiroffo, C. B. (2007). Poly(ADP-ribose) polymerase-1 (PARP-1) controls lung cell proliferation and repair after hyperoxia-induced lung damage. Am J Physiol Lung Cell Mol Physiol, 293(3), L619-629. doi: 10.1152/ajplung.00037.2007 Paglin, S., Hollister, T., Delohery, T., Hackett, N., McMahill, M., Sphicas, E., . . . Yahalom, J. (2001). A novel response of cancer cells to radiation Page 201 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer involves autophagy and formation of acidic vesicles. Cancer Res, 61(2), 439-444. Pavlidou, A., & Vlahos, N. F. (2014). Molecular alterations of PI3K/Akt/mTOR pathway: a therapeutic target in endometrial cancer. ScientificWorldJournal, 2014, 709736. doi: 10.1155/2014/709736 Pearce, L. R., Sommer, E. M., Sakamoto, K., Wullschleger, S., & Alessi, D. R. (2011). Protor-1 is required for efficient mTORC2-mediated activation of SGK1 in the kidney. Biochem J, 436(1), 169-179. doi: 10.1042/BJ20102103 Pearson, R. B., Dennis, P. B., Han, J. W., Williamson, N. A., Kozma, S. C., Wettenhall, R. E., & Thomas, G. (1995). The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain. EMBO J, 14(21), 5279-5287. Peponi, E., Drakos, E., Reyes, G., Leventaki, V., Rassidakis, G. Z., & Medeiros, L. J. (2006). Activation of mammalian target of rapamycin signaling promotes cell cycle progression and protects cells from apoptosis in mantle cell lymphoma. Am J Pathol, 169(6), 2171-2180. doi: 10.2353/ajpath.2006.051078 Peterson, R. T., Desai, B. N., Hardwick, J. S., & Schreiber, S. L. (1999). Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein. Proc Natl Acad Sci U S A, 96(8), 4438-4442. Peterson, T. R., Laplante, M., Thoreen, C. C., Sancak, Y., Kang, S. A., Kuehl, W. M., . . . Sabatini, D. M. (2009). DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell, 137(5), 873-886. doi: 10.1016/j.cell.2009.03.046 Pohl, G., Rudas, M., Taucher, S., Stranzl, T., Steger, G. G., Jakesz, R., . . . Filipits, M. (2003). Expression of cell cycle regulatory proteins in breast carcinomas before and after preoperative chemotherapy. Breast Cancer Res Treat, 78(1), 97-103. Polak, P., & Hall, M. N. (2009). mTOR and the control of whole body metabolism. Curr Opin Cell Biol, 21(2), 209-218. doi: 10.1016/j.ceb.2009.01.024 Page 202 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Populo, H., Lopes, J. M., & Soares, P. (2012). The mTOR Signalling Pathway in Human Cancer. Int J Mol Sci, 13(2), 1886-1918. doi: 10.3390/ijms13021886 Pourgholami, M. H., Cai, Z. Y., Badar, S., Wangoo, K., Poruchynsky, M. S., & Morris, D. L. (2010). Potent inhibition of tumoral hypoxia-inducible factor 1alpha by albendazole. BMC Cancer, 10, 143. doi: 10.1186/1471-2407- 10-143 Pourgholami, M. H., Yan Cai, Z., Lu, Y., Wang, L., & Morris, D. L. (2006). Albendazole: a potent inhibitor of vascular endothelial growth factor and malignant ascites formation in OVCAR-3 tumor-bearing nude mice. Clin Cancer Res, 12(6), 1928-1935. doi: 10.1158/1078-0432.CCR-05-1181 Puissant, A., Fenouille, N., & Auberger, P. (2012). When autophagy meets cancer through p62/SQSTM1. Am J Cancer Res, 2(4), 397-413. Pullen, N., & Thomas, G. (1997). The modular phosphorylation and activation of p70s6k. FEBS Lett, 410(1), 78-82. Rabinowitz, J. D., & White, E. (2010). Autophagy and metabolism. SCIENCE, 330(6009), 1344-1348. doi: 10.1126/science.1193497 Ramirez-Valle, F., Badura, M. L., Braunstein, S., Narasimhan, M., & Schneider, R. J. (2010). Mitotic raptor promotes mTORC1 activity, G(2)/M cell cycle progression, and internal ribosome entry site-mediated mRNA translation. Mol Cell Biol, 30(13), 3151-3164. doi: 10.1128/MCB.00322- 09 Rao, T. S., Correa, L. D., & Lloyd, G. K. (1997). Effects of lobeline and dimethylphenylpiperazinium iodide (DMPP) on N-methyl-D-aspartate (NMDA)-evoked acetylcholine release in vitro: evidence for a lack of involvement of classical neuronal nicotinic acetylcholine receptors. Neuropharmacology, 36(1), 39-50. Ravikumar, B., Vacher, C., Berger, Z., Davies, J. E., Luo, S., Oroz, L. G., . . . Rubinsztein, D. C. (2004). Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet, 36(6), 585-595. doi: 10.1038/ng1362 Page 203 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Ricciardelli, C., & Oehler, M. K. (2009). Diverse molecular pathways in ovarian cancer and their clinical significance. Maturitas, 62(3), 270-275. doi: 10.1016/j.maturitas.2009.01.001 Risch, H. A. (1998). Hormonal etiology of epithelial ovarian cancer, with a hypothesis concerning the role of androgens and progesterone. J Natl Cancer Inst, 90(23), 1774-1786. Robert, F., & Pelletier, J. (2009). Translation initiation: a critical signalling node in cancer. Expert Opin Ther Targets, 13(11), 1279-1293. doi: 10.1517/14728220903241625 Rosse, C., Linch, M., Kermorgant, S., Cameron, A. J., Boeckeler, K., & Parker, P. J. (2010). PKC and the control of localized signal dynamics. Nat Rev Mol Cell Biol, 11(2), 103-112. doi: 10.1038/nrm2847 Rouschop, K. M., & Wouters, B. G. (2009). Regulation of autophagy through multiple independent hypoxic signaling pathways. Curr Mol Med, 9(4), 417-424. Rufener, L., Baur, R., Kaminsky, R., Maser, P., & Sigel, E. (2010). Monepantel allosterically activates DEG-3/DES-2 channels of the gastrointestinal nematode Haemonchus contortus. Mol Pharmacol, 78(5), 895-902. doi: 10.1124/mol.110.066498 Rufener, L., Bedoni, N., Baur, R., Rey, S., Glauser, D. A., Bouvier, J., . . . Puoti, A. (2013). acr-23 Encodes a monepantel-sensitive channel in Caenorhabditis elegans. PLoS Pathog, 9(8), e1003524. doi: 10.1371/journal.ppat.1003524 Rufener, L., Keiser, J., Kaminsky, R., Maser, P., & Nilsson, D. (2010). Phylogenomics of ligand-gated ion channels predicts monepantel effect. PLoS Pathog, 6(9), e1001091. doi: 10.1371/journal.ppat.1001091 Ruiz-Lancheros, E., Viau, C., Walter, T. N., Francis, A., & Geary, T. G. (2011). Activity of novel nicotinic anthelmintics in cut preparations of Caenorhabditis elegans. Int J Parasitol, 41(3-4), 455-461. doi: 10.1016/j.ijpara.2010.11.009 Page 204 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Saci, A., Cantley, L. C., & Carpenter, C. L. (2011). Rac1 regulates the activity of mTORC1 and mTORC2 and controls cellular size. Mol Cell, 42(1), 50-61. doi: 10.1016/j.molcel.2011.03.017 Sadler, T. M., Gavriil, M., Annable, T., Frost, P., Greenberger, L. M., & Zhang, Y. (2006). Combination therapy for treating breast cancer using antiestrogen, ERA-923, and the mammalian target of rapamycin inhibitor, temsirolimus. Endocr Relat Cancer, 13(3), 863-873. doi: 10.1677/erc.1.01170 Samuels, Y., & Waldman, T. (2010). Oncogenic mutations of PIK3CA in human cancers. Curr Top Microbiol Immunol, 347, 21-41. doi: 10.1007/82_2010_68 Samuels, Y., Wang, Z., Bardelli, A., Silliman, N., Ptak, J., Szabo, S., . . . Velculescu, V. E. (2004). High frequency of mutations of the PIK3CA gene in human cancers. SCIENCE, 304(5670), 554. doi: 10.1126/science.1096502 Sancak, Y., Bar-Peled, L., Zoncu, R., Markhard, A. L., Nada, S., & Sabatini, D. M. (2010). Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell, 141(2), 290-303. doi: 10.1016/j.cell.2010.02.024 Sarbassov, D. D., Ali, S. M., Kim, D. H., Guertin, D. A., Latek, R. R., Erdjument- Bromage, H., . . . Sabatini, D. M. (2004). Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol, 14(14), 1296-1302. doi: 10.1016/j.cub.2004.06.054 Sarbassov, D. D., Ali, S. M., & Sabatini, D. M. (2005). Growing roles for the mTOR pathway. Curr Opin Cell Biol, 17(6), 596-603. doi: 10.1016/j.ceb.2005.09.009 Sarbassov, D. D., Ali, S. M., Sengupta, S., Sheen, J. H., Hsu, P. P., Bagley, A. F., . . . Sabatini, D. M. (2006). Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell, 22(2), 159-168. doi: 10.1016/j.molcel.2006.03.029 Satoo, K., Noda, N. N., Kumeta, H., Fujioka, Y., Mizushima, N., Ohsumi, Y., & Inagaki, F. (2009). The structure of Atg4B-LC3 complex reveals the Page 205 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer mechanism of LC3 processing and delipidation during autophagy. EMBO J, 28(9), 1341-1350. doi: 10.1038/emboj.2009.80 Sawyers, C. L. (2003). Will mTOR inhibitors make it as cancer drugs? Cancer Cell, 4(5), 343-348. Scarlatti, F., Granata, R., Meijer, A. J., & Codogno, P. (2009). Does autophagy have a license to kill mammalian cells? Cell Death Differ, 16(1), 12-20. doi: 10.1038/cdd.2008.101 Schmelzle, T., & Hall, M. N. (2000). TOR, a central controller of cell growth. Cell, 103(2), 253-262. Sehgal, S. N. (2003). Sirolimus: its discovery, biological properties, and mechanism of action. Transplant Proc, 35(3 Suppl), 7S-14S. Sengupta, S., Peterson, T. R., & Sabatini, D. M. (2010). Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell, 40(2), 310-322. doi: 10.1016/j.molcel.2010.09.026 Shapiro, G. I. (2006). Cyclin-dependent kinase pathways as targets for cancer treatment. J Clin Oncol, 24(11), 1770-1783. doi: 10.1200/JCO.2005.03.7689 Shaw, R. J., & Cantley, L. C. (2006). Ras, PI(3)K and mTOR signalling controls tumour cell growth. nature, 441(7092), 424-430. doi: 10.1038/nature04869 Shinjyo, T., Kuribara, R., Inukai, T., Hosoi, H., Kinoshita, T., Miyajima, A., . . . Inaba, T. (2001). Downregulation of Bim, a proapoptotic relative of Bcl-2, is a pivotal step in cytokine-initiated survival signaling in murine hematopoietic progenitors. Mol Cell Biol, 21(3), 854-864. doi: 10.1128/MCB.21.3.854-864.2001 Shintani, T., & Klionsky, D. J. (2004). Autophagy in health and disease: a double-edged sword. Science, 306(5698), 990-995. doi: 10.1126/science.1099993 Shiota, C., Woo, J. T., Lindner, J., Shelton, K. D., & Magnuson, M. A. (2006). Multiallelic disruption of the rictor gene in mice reveals that mTOR Page 206 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer complex 2 is essential for fetal growth and viability. Dev Cell, 11(4), 583- 589. doi: 10.1016/j.devcel.2006.08.013 Singh, R., Xiang, Y., Wang, Y., Baikati, K., Cuervo, A. M., Luu, Y. K., . . . Czaja, M. J. (2009). Autophagy regulates adipose mass and differentiation in mice. J Clin Invest, 119(11), 3329-3339. doi: 10.1172/JCI39228 Skripsky, T., & Hoffmann, S. (2010). Assessment of risk of monepantel faecal residues to dung fauna. Aust Vet J, 88(12), 490-496. doi: 10.1111/j.1751- 0813.2010.00645.x Sou, Y. S., Waguri, S., Iwata, J., Ueno, T., Fujimura, T., Hara, T., . . . Komatsu, M. (2008). The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice. Mol Biol Cell, 19(11), 4762-4775. doi: 10.1091/mbc.E08-03-0309 Soukas, A. A., Kane, E. A., Carr, C. E., Melo, J. A., & Ruvkun, G. (2009). Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans. Genes Dev, 23(4), 496-511. doi: 10.1101/gad.1775409 Sridharan, S., Jain, K., & Basu, A. (2011). Regulation of autophagy by kinases. Cancers (Basel), 3(2), 2630-2654. doi: 10.3390/cancers3022630 Stacey, D. W. (2003). Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells. Curr Opin Cell Biol, 15(2), 158-163. Stuchlikova, L., Jirasko, R., Vokral, I., Valat, M., Lamka, J., Szotakova, B., . . . Skalova, L. (2014). Metabolic pathways of anthelmintic drug monepantel in sheep and in its parasite (Haemonchus contortus). Drug Test Anal, 6(10), 1055-1062. doi: 10.1002/dta.1630 Su, B., & Jacinto, E. (2011). Mammalian TOR signaling to the AGC kinases. Crit Rev Biochem Mol Biol, 46(6), 527-547. doi: 10.3109/10409238.2011.618113 Tanaka, S., & Araki, H. (2010). Regulation of the initiation step of DNA replication by cyclin-dependent kinases. Chromosoma, 119(6), 565-574. doi: 10.1007/s00412-010-0291-8 Page 207 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Taylor, R. C., Cullen, S. P., & Martin, S. J. (2008). Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol, 9(3), 231-241. doi: 10.1038/nrm2312 Tee, A. R., Manning, B. D., Roux, P. P., Cantley, L. C., & Blenis, J. (2003). Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb. Curr Biol, 13(15), 1259-1268. Tirado, O. M., Mateo-Lozano, S., & Notario, V. (2005). Rapamycin induces apoptosis of JN-DSRCT-1 cells by increasing the Bax : Bcl-xL ratio through concurrent mechanisms dependent and independent of its mTOR inhibitory activity. Oncogene, 24(20), 3348-3357. doi: 10.1038/sj.onc.1208471 Torpy, J. M., Burke, A. E., & Golub, R. M. (2011). JAMA patient page. Ovarian cancer. JAMA, 305(23), 2484. doi: 10.1001/jama.305.23.2484 Towle, M. J., Salvato, K. A., Budrow, J., Wels, B. F., Kuznetsov, G., Aalfs, K. K., . . . Littlefield, B. A. (2001). In vitro and in vivo anticancer activities of synthetic macrocyclic ketone analogues of halichondrin B. Cancer Res, 61(3), 1013-1021. Trauner, M., Arrese, M., & Wagner, M. (2010). Fatty liver and lipotoxicity. Biochim Biophys Acta, 1801(3), 299-310. doi: 10.1016/j.bbalip.2009.10.007 Treinin, M., Gillo, B., Liebman, L., & Chalfie, M. (1998). Two functionally dependent acetylcholine subunits are encoded in a single Caenorhabditis elegans operon. Proc Natl Acad Sci U S A, 95(26), 15492-15495. Tritten, L., Silbereisen, A., & Keiser, J. (2011). In vitro and in vivo efficacy of Monepantel (AAD 1566) against laboratory models of human intestinal nematode infections. PLoS Negl Trop Dis, 5(12), e1457. doi: 10.1371/journal.pntd.0001457 Tsukamoto, S., Kuma, A., Murakami, M., Kishi, C., Yamamoto, A., & Mizushima, N. (2008). Autophagy is essential for preimplantation development of mouse embryos. Science, 321(5885), 117-120. doi: 10.1126/science.1154822 Page 208 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Twig, G., Hyde, B., & Shirihai, O. S. (2008). Mitochondrial fusion, fission and autophagy as a quality control axis: the bioenergetic view. Biochim Biophys Acta, 1777(9), 1092-1097. doi: 10.1016/j.bbabio.2008.05.001 Vander Heiden, M. G., Cantley, L. C., & Thompson, C. B. (2009). Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science, 324(5930), 1029-1033. doi: 10.1126/science.1160809 Vermeulen, K., Van Bockstaele, D. R., & Berneman, Z. N. (2003). The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif, 36(3), 131-149. Vicencio, J. M., Ortiz, C., Criollo, A., Jones, A. W., Kepp, O., Galluzzi, L., . . . Kroemer, G. (2009). The inositol 1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1. Cell Death Differ, 16(7), 1006-1017. doi: 10.1038/cdd.2009.34 Vichai, V., & Kirtikara, K. (2006). Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc, 1(3), 1112-1116. doi: 10.1038/nprot.2006.179 Vignot, S., Faivre, S., Aguirre, D., & Raymond, E. (2005). mTOR-targeted therapy of cancer with rapamycin derivatives. Ann Oncol, 16(4), 525-537. doi: 10.1093/annonc/mdi113 Waller, P. J., Rudby-Martin, L., Ljungstrom, B. L., & Rydzik, A. (2004). The epidemiology of abomasal nematodes of sheep in Sweden, with particular reference to over-winter survival strategies. Vet Parasitol, 122(3), 207-220. doi: 10.1016/j.vetpar.2004.04.007 Wan, X., & Helman, L. J. (2007). The biology behind mTOR inhibition in sarcoma. Oncologist, 12(8), 1007-1018. doi: 10.1634/theoncologist.12-8- 1007 Wang, C. Y., Kim, H. H., Hiroi, Y., Sawada, N., Salomone, S., Benjamin, L. E., . . . Liao, J. K. (2009). Obesity increases vascular senescence and susceptibility to ischemic injury through chronic activation of Akt and mTOR. Sci Signal, 2(62), ra11. doi: 10.1126/scisignal.2000143 Page 209 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Wang, R. H., Kim, H. S., Xiao, C., Xu, X., Gavrilova, O., & Deng, C. X. (2011). Hepatic Sirt1 deficiency in mice impairs mTorc2/Akt signaling and results in hyperglycemia, oxidative damage, and insulin resistance. J Clin Invest, 121(11), 4477-4490. doi: 10.1172/JCI46243 Watanabe, R., Wei, L., & Huang, J. (2011). mTOR signaling, function, novel inhibitors, and therapeutic targets. J Nucl Med, 52(4), 497-500. doi: 10.2967/jnumed.111.089623 Wen, F., He, S., Sun, C., Li, T., & Wu, S. (2014). PIK3CA and PIK3CB expression and relationship with multidrug resistance in colorectal carcinoma. Int J Clin Exp Pathol, 7(11), 8295-8303. Wendel, H. G., De Stanchina, E., Fridman, J. S., Malina, A., Ray, S., Kogan, S., . . . Lowe, S. W. (2004). Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy. nature, 428(6980), 332-337. doi: 10.1038/nature02369 Wesierska-Gadek, J., & Schmid, G. (2001). Poly(ADP-ribose) polymerase-1 regulates the stability of the wild-type p53 protein. Cell Mol Biol Lett, 6(2), 117-140. White, E. (2014). Q&A: targeting autophagy in cancer-a new therapeutic? Cancer Metab, 2, 14. doi: 10.1186/2049-3002-2-14 White, E., & DiPaola, R. S. (2009). The double-edged sword of autophagy modulation in cancer. Clin Cancer Res, 15(17), 5308-5316. doi: 10.1158/1078-0432.CCR-07-5023 Wise, D. R., DeBerardinis, R. J., Mancuso, A., Sayed, N., Zhang, X. Y., Pfeiffer, H. K., . . . Thompson, C. B. (2008). Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proc Natl Acad Sci U S A, 105(48), 18782-18787. doi: 10.1073/pnas.0810199105 Wu, Y. T., Tan, H. L., Shui, G., Bauvy, C., Huang, Q., Wenk, M. R., . . . Shen, H. M. (2010). Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J Biol Chem, 285(14), 10850-10861. doi: 10.1074/jbc.M109.080796 Page 210 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Xie, X., White, E. P., & Mehnert, J. M. (2013). Coordinate autophagy and mTOR pathway inhibition enhances cell death in melanoma. PLoS One, 8(1), e55096. doi: 10.1371/journal.pone.0055096 Xu, Y., Luo, Q., Lin, T., Zeng, Z., Wang, G., Zeng, D., . . . Chen, H. (2014). U12, a UDCA derivative, acts as an anti-hepatoma drug lead and inhibits the mTOR/S6K1 and cyclin/CDK complex pathways. PLoS One, 9(12), e113479. doi: 10.1371/journal.pone.0113479 Yang, H., Rudge, D. G., Koos, J. D., Vaidialingam, B., Yang, H. J., & Pavletich, N. P. (2013). mTOR kinase structure, mechanism and regulation. Nature, 497(7448), 217-223. doi: 10.1038/nature12122 Yang, Q., & Guan, K. L. (2007). Expanding mTOR signaling. Cell Res, 17(8), 666-681. doi: 10.1038/cr.2007.64 Yang, S., Wang, X., Contino, G., Liesa, M., Sahin, E., Ying, H., . . . Kimmelman, A. C. (2011). Pancreatic cancers require autophagy for tumor growth. Genes Dev, 25(7), 717-729. doi: 10.1101/gad.2016111 Yang, Z., & Klionsky, D. J. (2010). Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol, 22(2), 124-131. doi: 10.1016/j.ceb.2009.11.014 Yang, Z. J., Chee, C. E., Huang, S., & Sinicrope, F. A. (2011). The role of autophagy in cancer: therapeutic implications. Mol Cancer Ther, 10(9), 1533-1541. doi: 10.1158/1535-7163.MCT-11-0047 Yassin, L., Gillo, B., Kahan, T., Halevi, S., Eshel, M., & Treinin, M. (2001). Characterization of the deg-3/des-2 receptor: a nicotinic acetylcholine receptor that mutates to cause neuronal degeneration. Mol Cell Neurosci, 17(3), 589-599. doi: 10.1006/mcne.2000.0944 Young, A. R., Narita, M., Ferreira, M., Kirschner, K., Sadaie, M., Darot, J. F., . . . Narita, M. (2009). Autophagy mediates the mitotic senescence transition. Genes Dev, 23(7), 798-803. doi: 10.1101/gad.519709 Yu, S., Shen, G., Khor, T. O., Kim, J. H., & Kong, A. N. (2008). Curcumin inhibits Akt/mammalian target of rapamycin signaling through protein Page 211 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer phosphatase-dependent mechanism. Mol Cancer Ther, 7(9), 2609-2620. doi: 10.1158/1535-7163.MCT-07-2400 Yuan, T. L., & Cantley, L. C. (2008). PI3K pathway alterations in cancer: variations on a theme. Oncogene, 27(41), 5497-5510. doi: 10.1038/onc.2008.245 Zhang, F., Zhang, X., Li, M., Chen, P., Zhang, B., Guo, H., . . . Zhang, N. (2010). mTOR complex component Rictor interacts with PKCzeta and regulates cancer cell metastasis. Cancer Res, 70(22), 9360-9370. doi: 10.1158/0008-5472.CAN-10-0207 Zhang, J. F., Lu, M. Q., Cai, C. J., Yang, Y., Li, H., Yi, H. M., & Chen, G. H. (2006). [The role of caspase-3 in rapamycin-induced apoptosis of hepatocellular carcinoma BEL-7402 cells]. Ai Zheng, 25(12), 1508-1511. Zhong, Y., Wang, Q. J., Li, X., Yan, Y., Backer, J. M., Chait, B. T., . . . Yue, Z. (2009). Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex. Nat Cell Biol, 11(4), 468-476. doi: 10.1038/ncb1854 Zinzalla, V., Stracka, D., Oppliger, W., & Hall, M. N. (2011). Activation of mTORC2 by association with the ribosome. Cell, 144(5), 757-768. doi: 10.1016/j.cell.2011.02.014 References style: APA based on UNSW, medicine recommendation. http://web.med.unsw.edu.au/infoskills/apa/apa_print.htm Page 212