An analysis of the effects of CARP-1/CCAR1 functional mimetics on drug efflux transporters A thesis submitted to The University of Manchester for the degree of Master of Philosophy in the Faculty of Biology, Medicine and Health 2019 Shelley R Morris School of Health Sciences Division of Pharmacy and Optometry List of Contents List of Contents ............................................................................................................................. 2 List of Tables ................................................................................................................................. 7 List of Figures ................................................................................................................................ 8 List of Abbreviations ................................................................................................................... 10 Abstract ....................................................................................................................................... 13 Declaration .................................................................................................................................. 14 Copyright Statement ................................................................................................................... 15 Acknowledgements ..................................................................................................................... 16 The Author .................................................................................................................................. 17 1 Introduction ........................................................................................................................ 18 1.1 Definition and hallmarks of cancer ............................................................................. 18 1.2 Neuroblastoma and current treatments .................................................................... 19 1.3 CNS metastases – the requirement of novel treatments for neuroblastoma to cross the blood-brain barrier ........................................................................................................... 23 1.4 Characteristics of the blood-brain barrier .................................................................. 23 1.5 In vitro models of the BBB .......................................................................................... 27 1.6 SH-SY5Y cell line as an in vitro model of neuroblastoma............................................ 29 1.7 Multidrug resistance and prognostic implications for ABCB1, ABCG2 and ABCC in neuroblastoma ........................................................................................................................ 30 1.7.1 ABCB1 .................................................................................................................. 30 1.7.2 ABCG2 ................................................................................................................. 31 1.7.3 ABCC .................................................................................................................... 32 1.8 Limitations of current approaches to circumvent chemoresistance and increase BBB penetration ............................................................................................................................. 33 1.8.1 ABCB1 Inhibitors ................................................................................................. 33 1.8.2 ABCG2 Inhibitors ................................................................................................. 35 1.8.3 ABCC Inhibitors ................................................................................................... 36 1.8.4 Chemotherapeutics with dual ABC transporter inhibitory properties ............... 37 1.8.5 Modulators of ABC transporter expression ........................................................ 38 1.9 Novel approach to the cytotoxic treatment of neuroblastoma-derived CNS metastases and modulation of ABC transporters based on CARP-1/CCAR1 .......................... 38 1.9.1 Validation of CARP-1 as a drug target ................................................................. 38 1.9.2 Discovery of CARP-1 functional mimetics (CFMs) based on disruption of the CARP-1/Anaphase-promoting complex (APC)-2 interaction .............................................. 39 1.9.3 CFM-mediated upregulation of CARP-1 signalling in a range of cancer cell types 40 2 1.9.4 CFM-mediated cytotoxicity ................................................................................. 41 1.9.5 Additional mechanistic evidence supporting the proposed use of CFM-4.16 and CFM-4.17 in neuroblastoma ............................................................................................... 41 1.9.6 Effects of CFMs on signalling molecules implicated in the regulation of ABCB1 and ABCG2 activity and expression .................................................................................... 42 1.10 Aims and objectives .................................................................................................... 43 1.10.1 Aims ..................................................................................................................... 43 1.10.2 Objectives ............................................................................................................ 45 2 Methods .............................................................................................................................. 46 2.1 Reagents and materials ............................................................................................... 46 2.2 Isolation of porcine brain endothelial cells ................................................................. 47 2.3 Coating of PBEC culture surfaces ................................................................................ 48 2.4 Culture of PBECs .......................................................................................................... 48 2.5 Subculture of PBECs from 6-well plates onto 96-well plates ...................................... 48 2.6 Subculture of PBECs from 6-well plates onto Transwell® inserts ............................... 49 2.7 Trypan blue assay ........................................................................................................ 49 2.8 Preparation of CFM-4.16 and CFM-4.17 ..................................................................... 49 2.9 Culture of CTX-TNA2 astrocytes and production of astrocyte-conditioned medium . 50 2.10 Cryopreservation of CTX-TNA2 astrocytes .................................................................. 50 2.11 Subculture of CTX-TNA2 astrocytes onto 12-well plates ............................................ 50 2.12 Culture, cryopreservation and subculture of SH-SY5Y neuroblastoma cells .............. 51 2.13 Measurement of the effect of CFM-4.16 and CFM-4.17 on SH-SY5Y viability using the MTT assay................................................................................................................................ 51 2.14 Measurement of the effect of CFM-4.16 and CFM-4.17 on PBEC viability using the neutral red assay ..................................................................................................................... 52 2.15 Measurement of the effect of ABCB1 and ABCG2 inhibitors on CFM-4.16 and CFM- 4.17–mediated toxicity in PBECs............................................................................................. 53 2.16 Measurement of ABC transporter functional activity in SH-SY5Y cells and in porcine brain endothelial cells ............................................................................................................. 53 2.17 Measurement of ABCB1 transporter functional activity ............................................ 53 2.18 Measurement of ABCG2 transporter functional activity ............................................ 55 2.19 Measurement of ABCC transporter functional activity .............................................. 56 2.20 Measurement of cellular protein content .................................................................. 57 2.21 Generating the in vitro blood-brain barrier model ..................................................... 57 2.22 Measurement of transendothelial electrical resistance ............................................. 58 2.23 Measurement of the apparent permeability of PBEC monolayers to Lucifer yellow . 58 2.24 Determination of absorption characteristics of CFM-4.16 and CFM-4.17 ................. 59 2.25 Measurement of the penetration of CFM-4.16 and CFM-4.17 across an in vitro model of the blood-brain barrier ....................................................................................................... 60 3 2.26 Indirect measurement of the permeability of CFM-4.16 and CFM-4.17 across an in vitro model of the blood-brain barrier.................................................................................... 60 2.27 Statistical analysis ....................................................................................................... 61 3 Results 1 (Neuroblastoma cell viability and functional assays) .......................................... 62 3.1 Visualisation of the morphology of SH-SY5Y cells ....................................................... 62 3.2 Evaluation of the effect of CFM-4.16 and CFM-4.17 on the viability of SH-SY5Y cells 62 3.3 Determination of ABCB1 functional activity in SH-SY5Y