Sensitivity to and functional effects of tricyclic agents on glioma: an immunohistochemical and in vitro study Thesis Submitted By: KATHARINE ANNE PEREGRÍN BSc This thesis is submitted in partial fulfilment of the requirements for the award of the degree of Doctor of Philosophy of the University of Portsmouth Cellular and Molecular Neuro-oncology Research Group University of Portsmouth June 2011 I Abstract The current outlook for patients suffering malignant glioma is poor, with sub- optimal delivery of agents across the blood-brain barrier, migration of tumour cells to areas that are not exposed to chemotherapeutic assault and the ability of tumour cells to repair DNA damage caused by anti-proliferative agents. The objectives were to demonstrate the potential of targeting the mitochondria of glioma cells through the use of tricyclic antidepressants and to show that the success of this approach is largely dependent on the metabolic capacity of the patient and the expression of the norepinephrine transporter on glioma cells. A range of tricyclic agents were screened against a panel of tumours using MTT, ATP-TCA and Annexin-V assays; the metabolic capacity of seventeen glioma patients was assessed through HPLC-MS/MS sampling of plasma Clomipramine concentrations and cytochrome P450 drug metabolism enzyme genotyping via real-time PCR; the presence of the norepinephrine transporter was elucidated through immunohistochemical, immunocytochemical, Western blotting and real- time PCR techniques and finally apoptotic potential was determined by screening tumours (retrospectively and prospectively) via real-time PCR. Chemosensitivity results show that Clomipramine (the range for five tumours tested was 42.57 ± 16.58 µM) and Nortriptyline (the range for four tumours tested was 30.22 ± 14.81 µM) were the most effective agents when tested in the MTT assay and Norclomipramine (the range for five tumours tested was 7.65 ±3.53 µM) and Nortriptyline (the range for five tumours tested was 33.15 ± 13.72 µM) were the most effective agents when tested using the ATP-TCA assay. Annexin-V flow cytometry supported these results and further evidenced that Clomipramine induces apoptosis in malignant glioma. II The genotypic status of CYP2D6 and CYP2C19, combined with plasma levels of Clomipramine/Norclomipramine achieved in vivo, showed that mutations in the CYP2D6*2X3 allele significantly affect the metabolism of Norclomipramine (p<0.05). Immunohistochemical, immunocytochemical and Western blotting techniques demonstrated the presence of the norepinephrine transporter (encoded by the SLC6A2 gene) on glioma cells, however real-time PCR results suggest that the exons within the SLC6A2 gene contain splice variants. Taqman low density array of retrospective tumour samples revealed variation in the expression of apoptotic genes, with no discernable pattern, and that it is possible to modulate the expression of these genes by exposing SNB-19, DK-MG and UPAB glioma cells to tricyclic antidepressants, Procarbazine, Dexamethasone and Valproic acid. Evidence from this study demonstrates that tricyclic antidepressants provide a new approach to mitochondrially-mediated therapy for malignant glioma that express the Norepinephrine transporter, which overcomes the resistance to targeting proliferation and growth factors. III II Acknowledgements Firstly I wish to thank Professor Geoffrey Pilkington. I would not have had such a fantastic project had it not been for Geoff‟s wealth of knowledge in the subject area, years of experience, kindness towards our patients and most importantly emphasis on working hard and playing hard. Secondly I wish to thank Professor Ian Cree and the members of the TORC Laboratory for their support. Thank you to the Samantha Dickson Brain Tumour Trust, not only for part funding my salary but for their help in liaising with patients during this study. Special thanks go to my wonderful parents for their energy, optimism and intelligence. They have been a constant emotional, moral and of course financial support for nearly thirty years and this PhD would not exist without them. Finally to my husband Michael and son Thomas, this is for you. IV III Declaration I declare that whilst studying for the degree of Doctorate of Philosophy at the University of Portsmouth I have not been registered for any other award at another University. The work undertaken for this degree has not been submitted elsewhere for any other award. The work contained in this thesis is my own with the following exceptions: The immunohistochemistry was performed by Mrs P. Johnson The HPLC-MS/MS was performed by Dr Richard Walker. The MTT assay using IPSB-18 was performed by Mr T. Ahmed. V 1 General Introduction ...................................................................................... 17 1.1 Background to Brain Tumours .................................................................. 18 1.2 Classification and Grading of brain tumours ............................................ 20 1.3 Pathology and Genetics of tumour types included in this study ............... 23 1.3.1 Astrocytic Tumours .............................................................................. 23 1.3.2 Glioblastoma multiforme (WHO grade IV) ......................................... 25 1.3.3 Oligodendroglioma .............................................................................. 25 1.3.4 Meningioma ......................................................................................... 26 1.3.5 Paediatric medulloblastoma and supratentorial primitive neuroectodermal tumours ..................................................................................... 26 1.4 Biology of brain tumours .......................................................................... 27 1.4.1 Glioma angiogenesis and Vascular Endothelial Growth Factor-A ...... 29 1.5 The Blood-Brain Barrier ........................................................................... 31 1.6 Tried and tested approaches for malignant glioma ................................... 33 1.7 Traditional chemotherapy targets glioma cell proliferation ...................... 34 1.7.1 Nitrosoureas agents .............................................................................. 35 1.7.2 Procarbazine, CCNU (Lomustine) and Vincristine (PCV) .................. 36 1.7.3 Topoisomerase inhibitors ..................................................................... 38 1.7.4 Platinum analogues .............................................................................. 40 1.7.5 The structures of traditional glioma chemotherapy agents that target proliferation .......................................................................................................... 42 1.8 Recent approaches that have shown modest results .................................. 43 1.8.1 An oral agent to target proliferation ..................................................... 43 1.8.2 Targeting the platelet-derived growth factor receptor ......................... 44 1.8.3 Bypassing the blood-brain barrier with impregnated wafers ............... 45 1.8.4 Blocking the vascular endothelial growth factor receptor with a monoclonal antibody ............................................................................................ 46 1.8.5 Modulating the immune response to tumours with antibodies and cytokines 46 1.8.6 Delivering chemotherapy directly to the tumour site ........................... 49 1.8.7 Reprogramming glioma cells with gene therapy ................................. 51 1.9 Approaching the mitochondrion as a new target for malignant glioma .... 56 1.10 The role of mitochondria in determining the fate of glioma cells............. 57 VI 1.11 The generation of adensine triphosphate provides a selective target in tumour cells .......................................................................................................... 63 1.12 Oxygen sensing by mitochondria at complex III ...................................... 64 1.13 Tricyclic agents with known Blood-Brain Barrier penetration as candidates for targeting mitochondria .................................................................. 67 1.13.1 Antidepressants have an affinity for the serotonin and norepinephrine transporters ........................................................................................................... 67 1.13.2 Other tricyclic compounds in clinical use that may show cytotoxic activity against malignant glioma ........................................................................ 70 1.14 Clomipramine hydrochloride: a tricyclic antidepressant with the potential to selectively trigger apoptosis in tumour cells .................................................... 71 1.15 Drawbacks when selecting tricyclic antidepressants for use in patients with malignant glioma.......................................................................................... 74 1.16 The norepinephrine transporter (NET): the link between tricyclic antidepressants and apoptosis in glioma? ............................................................ 76 1.17 Apoptosis .................................................................................................. 78 1.18 The Mitochondrial Permeability Transition Pore in apoptosis ................. 80 1.19 Mitochondrial Outer Membrane Permeabilisation in apoptosis ............... 81 1.20 Extrinsic and intrinsic pathways of apoptosis ........................................... 82 1.21 The role of inhibitor of apoptosis-inducing proteins