Nghiên cứu hiệu quả của Aspirin và tương tự trong điều trị ung thư não

Assessment of the in vitro efficacy of aspirin and aspirin analogues in combination with standard chemotherapeutics in glioma cell lines by Preet Chheda A thesis submitted in partial fulfilment for the requirements for the degree of Masters of Science by Research at the University of Central Lancashire September 2014 1 STUDENT DECLARATION FORM Concurrent registration for two or more academic awards I declare that while registered as a candidate for the research degree, I have not been a registered candidate or enrolled student for another award of the University or other academic or professional institution ______________________________________________________________________ Material submitted for another award I declare that no material contained in the thesis has been used in any other submission for an academic award and is solely my own work ______________________________________________________________________ Collaboration Where a candidate’s research programme is part of a collaborative project, the thesis must indicate in addition clearly the candidate’s individual contribution and the extent of the collaboration. Please state below: Signature of Candidate ________________________________________________ Type of Award _________________________________________________ School _________________________________________________ i Abstract Glioblastoma multiforme (GBM) is the most common and most malignant primary brain tumour. The prognosis for patients remains poor with a median survival time falling well short of a year in spite of surgical removal of tumour followed by radiotherapy. GBM is resistant to the current treatment regimen, often due to therapeutic resistance, however combination therapies have been proven to have a beneficial effect on the prognosis of patients.

Aspirin, one of the most commonly used drugs reduces the viability and proliferation of glioblastoma both in vitro and in vivo. This study examined the combination of aspirin or its analogue PN517 with the standard chemotherapeutic (cisplatin) and determined effects on cell viability, apoptosis and caspase activity. Either aspirin or PN517 combined with cisplatin significantly reduced the viability of U87-MG glioblastoma cells. The apoptosis induced by cisplatin was augmented in combination with either PN517 or aspirin.

These results for the first time suggest a potential for improved treatment of glioblastoma multiforme where aspirin or its analogue PN517 are combined with the standard chemotherapeutic drugs cisplatin. ii Table of Contents 1 Introduction .1 Introduction – Central Nervous System Tumours .2 Classification and Grading of Central Nervous System Tumours .4 Glioma Classification and Grading .5 Molecular Markers of Glioma .8 Diagnosis of Glioma .9 Treatment of Glioma .2 Chemotherapy & Alkylating Agents .10 Non-steroidal Anti-inflammatory Drugs .2 Aspirin and Cancer.3 Aspirin and Metastasis .4 Aspirin and Glioma .15 Hypothesis and Aims.33 2 Materials and Methods.2 Cell Culture Methods .3 PrestoBlue® Cell Viability Assay .4 CFDA-SE Cell Proliferation Assay .5 Apoptosis Assay, Annexin-V/Propidium Iodide .7 Assay of Caspase 8 & 9 Activity .8 Confocal Laser Scanning Microscopy .1 Increasing Cell Number Assays .2 Concentration Response Assays .3 Combination Treatment Assays - Simultaneous .4 Combination Treatment Assays - Staggered .5 Combination Treatment Assays - Multiple .1 Annexin-V and Propidium Iodide .2 Caspase 8 and 9 Activation .3 Laser Scanning Confocal Microscopy .1 Growth Curve and PrestoBlue® Linearity .4 Apoptosis and Autophagy .99 v Table of figures: Figure 1.1: Chemical structure of aspirin .2: Chemical structure of PN517 .3: Schematic representation of the intrinsic and extrinsic apoptosis pathways 29 Figure 1.4: Schematic representation of the PI3K/mTOR signalling pathways in autophagy .1: Schematic diagram depicting the Caspase-Glo assay protocol .1: Growth curve for U-87 MG and SVG-p12 cell lines.2: Effect of increasing cell number on fluorescence determined by PrestoBlue® assay in U-87 MG and SVG-p12 cell lines.3: Data illustrating the effect of dose-dependent treatment of aspirin (closed circle) and cisplatin (closed square) on U87-MG and SVG-P12 cell lines.4: Dose dependent effects of temozolomide (closed circle) and PN517 (closed square) on cell viability in U87-MG and SVG-P12 over 24 and 48 hours.5: Cell viability following combination treatment of aspirin and PN517 with cisplatin and of aspirin with PN517 in a time dependent manner in U87-MG cell line.6: Cell viability following combination treatment of aspirin and PN517 with cisplatin and of aspirin with PN517 in a time dependent manner in SVG-P12 cell line.7: Cell viability after staggered treatment of aspirin/PN517 followed by cisplatin.8: Cell viability after prolonged treatment with aspirin/ analogue followed by cisplatin.9: Cell proliferation following drug treatment in U87-MG cell line.10: Cell proliferation following drug treatment in SVG-P12 cell line.11: Cell proliferation following drug treatment in U87-MG cell line.12: Cell proliferation following drug treatment in SVG-P12 cell line .13: Apoptosis following drug treatment in U87-MG cell line.14: Apoptosis following drug treatment in SVG-P12 cell line.15: Dot-plots for Apoptosis assay following 24 hours of drug treatment in both the cell lines.16: Dot-plots for Apoptosis assay following 48 hours of drug treatment in both the cell lines.17: Caspase-8 activity seen U87-MG and SVG-P12 cell lines.18: Caspase-9 activity seen U87-MG and SVG-P12 cell lines.19: Cell images of control treatments in U87-MG and SVG-P12.20: Cell images following drug treatment in U87 and SVG cells at 24 hours.21: Cell images following drug treatment in U87 and SVG cells at 48 hours.22: Confocal images showing induction of apoptosis following drug treatment in both the cell lines.23: Confocal images showing induction of apoptosis following drug treatment in both the cell lines.24: 3-MA dose response curve for U87-MG and SVG-P12 cell lines at 24 and 48 hours. 85 List of tables Table 1.1: WHO classification of CNS tumours (adapted from Louis et al.2: WHO classification of Gliomas (Louis et al.1: IC50 values for U-87 MG cell line following 24 and 48 hours incubation with aspirin, cisplatin and PN517.2: IC50 values for SVG-P12 cell line following 24 and 48 hours incubation with aspirin, cisplatin and PN517. 65 Acknowledgements There are many people to whom I owe a big gratitude for helping me through the course of this Masters by Research degree.

Firstly I’d like to thank my parents Dr. Bharat Chheda and Usha Chheda for their financial and emotional support. Their immovable support has helped me throughout the way and their encouragement has got me through tough times. I would like to express my deepest gratitude to my supervisor, Dr.

Philip Welsby, for his excellent guidance, caring, patience, and providing me with an excellent atmosphere for doing research. With his help I’ve learnt several new lab techniques and also improvised on several others. Without his invaluable guidance and persistent help this thesis would not have been possible. I deeply appreciate him being tolerant and supportive while I’ve written the thesis.

I am deeply grateful to Dr. Jaipaul Singh for his advice and guidance which has been of great help throughout my research. I would like to offer my special thanks to Dr. Gail Welsby for her expertise and helping me out with confocal microscopy.

viii I would like to thank Dr Julie Shorrocks for providing me with assistance in cell culture laboratory whenever it was needed. A special thanks to Flourina Thakor for her constant emotional support and encouragement. Abbreviations 3-MA 3-methyladenine 4HBZ 4-hydroxy benzoate zinc AMPK Adenosine monophosphate activated protein kinase ANOVA Analysis of variance CDDP Cisplatin CFDA-SE Carboxyfluorescein diacetate succinimidyl ester CFSE Carboxyfluorescein succinimidyl ester CNS Central Nervous System COX Cyclooxygenase CRC Colorectal cancer CT Computerised tomography CYP450 Cytochrome P450 DISC Death Inducing Signal Complex DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid EMEM Eagles Minimum Essential Medium FACS Fluorescence Activated Cell Sorting FBS Foetal Bovine Serum ix FITC Fluorescein isothiocyanate FS Forward Scatter GBM Glioblastoma Multiforme GSH Glutathione GY Gray Units IC50 Inhibitory concentration 50% IDH1 Isocitrate dehydrogenase 1 LC-MS Liquid chromatography-mass spectroscopy LSM Laser Scanning Microscope MDC Monodansylcadaverine MGMT O6-alkylguanine DNA alkyltransferase MMR Mismatch Repair MRI Magnetic Resonance Imaging MTIC 3-methyl-(triazen-1-yl)imidazole-4-carboxamide MTOR Mammalian target of rapamycin NEAA Non-essential amino acids NER Nucleotide Excision Repair NICE National Institute for Health and Clinical Excellence NSAID Non-steroidal anti-inflammatory drug PARP Poly (ADP-ribose) polymerase PBS Phosphate Buffered Saline PI Propidium iodide PI3K Phosphatidylinositol-4,5-bisphosphate 3-kinase SEM Standard error of mean SS Side Scatter x Stat3 Signal transducer and activator of transcription 3 TMZ Temozolomide TNF Tumour Necrosis Factor WHO World Health Organisation xi 1 Introduction 1 1.1 Introduction – Central Nervous System Tumours Brain tumours, while rare in comparison to cancers such as breast or prostate, represent a significant challenge both in terms of diagnosis and treatment. The UK annual incidence rate of brain tumours ranges from 14.8 per 100,000 for males and 14.6 per 100,000 for females (Cancer Research UK, 2014).

Primary brain tumours are those arising within the central nervous system (CNS) and represent 1.6% of all the tumours diagnosed, whereas secondary brain tumours, resulting from metastasis of non CNS tumours, represent 6% of all tumours detected in the UK (Cancer Research UK, 2014). While low grade tumours that are slow growing with well-defined borders can usually be removed surgically, their location within the CNS presents challenges due to the risk of significant neurological sequelae (Veeravagu et al. This risk increases with high grade tumours that grow rapidly and are highly invasive, but most studies show a significant improvement in survival that correlates the extent of tumour resection (Hervey-Jumper & Berger, 2014). The main cause of brain tumours, like several other cancers still remains unidentified.

However, several risk factors have been established which include exposure to radiation, previous incidence of cancer, genetic conditions such as neurofibromatosis, and other medical conditions such as AIDS (Reilly, 2010). Age is also a major factor as the risk of acquiring the disease increases with age, with the age group of 50-70 being the most affected (Eheman et al. Improvements in diagnosis, treatment, and prevention of cancer has resulted in a significant improvement in the median survival time for all cancers following diagnosis, from 1 year in 1971-72 to 5.8 years in 2007, with the biggest increase being in colon cancer, improving from 0. However, brain tumour survival time in UK has 2 shown little improvement, increasing from 0.3 years in 1971 to 0.

This report emphasises the significant challenges that remain in the diagnosis and treatment of brain tumours, and highlights the importance of the development of new treatment regimens for brain tumours.2 Classification and Grading of Central Nervous System Tumours Classification and grading is used to design individual patient treatment protocols and to determine their likely prognosis. Additionally, it creates a system that can be accepted worldwide allowing for global clinical trials and epidemiological studies (Louis et al. In 2007, the World Health Organisation (WHO) ratified a new wide-ranging classification of neoplasms affecting the CNS (Louis et al. The classification of brain tumours is based on the abnormal growth of a specific cell type, and grading helps in understanding the extent of malignancy or aggressiveness of the tumour, with higher grade corresponding to higher malignancy (Loukopoulos & Robinson, 2007).

The new WHO system is particularly useful in this regard with only a few notable exceptions for example, either all or almost all gemistocytic astrocytomas are actually anaplastic and hence named grade III or even IV rather than grade II. The WHO classification also provides a corresponding grading system for each type of tumour where most named tumours are of a single defined grade. An outline of this classification is provided below (Table 1.1: WHO classification of CNS tumours (adapted from Louis et al., 2007) Tumours of Neuroepithelial Tissue Astrocytic Tumours Choroid plexus Tumours Oligodendroglial Tumours Neuronal & mixed neuronal-glial tumours Oligoastrocytic Tumours Tumours of the pineal region Ependymal Tumours Embryonal tumours Other neuroepithelial tumours Tumours of Cranial and Paraspinal Nerves Schwannoma Perineuroma Neurofibroma Malignant peripheral nerve sheath tumour Tumours of the Meninges Tumours of melingothelial cells Primary melanocytic lesions Mesenchymal tumours Other neoplasms related to the meninges Lymphomas and Haematopoietic Neoplasm Malignant lymphomas Granulocytic sarcoma Plasmacytoma Germ Cell Tumours Germinoma Choriocarcinoma Embryonal carcinoma Teratoma Yolk sac tumour Mixed germ cell tumour Tumours of the Sellar Region Craniopharyngoma Pituicytoma Granular cell tumour Spindle cell oncocytoma he Meninges 4 Tumour grading plays a very important role in the choice of therapy, especially while ascertaining the adjuvant radiotherapy and chemotherapy protocols (NICE, 2006; Araujo et al. The grading of CNS tumours by WHO involves a scheme of grading that serves as a malignancy scale (Bratasz et al.