Date of Award
PhD (Doctor of Philosophy)
Professor Jochen Prehn
Mr Donncha O'Brien
Apoptosis, Inhibitor-of-Apoptosis-Protein Antagonist, Glioblastoma, Birinapant, Temozolomide
Resistance to temozolomide (TMZ) greatly limits chemotherapeutic effectiveness in glioblastoma (GBM). Within the currently presented thesis, we analysed the ability of the Inhibitor-of-apoptosis-protein (IAP) antagonist Birinapant to enhance treatment responses to TMZ in a panel of commercially available and patient-derived GBM cell lines. Responses to TMZ and Birinapant were analysed using colorimetric viability assays, flow cytometry, morphological analysis and protein expression profiling of anti- and pro- apoptotic proteins involve in intrinsic and extrinsic pathways. Cell death analysis identified three principal response patterns: Type A cells that readily activated caspase-8 and cell death in response to TMZ with minimal sensitisation following addition of Birinapant; Type B cells that readily activated caspase-8 and cell death in response to Birinapant but did not show further sensitization with TMZ; and Type C cells that showed no significant cell death or moderately enhanced cell death in the combined treatment paradigm.
Next, using the system biology model of effector caspase activation (APOPTO-CELL), we implemented the concentrations of five key apoptotic proteins (XIAP, procaspase-3, procaspase-9, Smac and Apaf-1), as analysed from western blot, into the model. The model’s output is identified as substrate cleavage of caspase-3 over time. Findings indicate APOPTO-CELL is a predictive tool of TMZ response in vitro, but no correlation were found between TMZ plus Birinapant treatment in vitro and APOPTO-CELL output following addition of IAP antagonist, suggesting that other pathway might be activated independent of MOMP-dependent caspase-3 activation. When comparing primary and recurrent patient tumour samples, significantly increased levels of procaspase-3 were found in primary tumour samples. The APOPTO-CELL model was able to predict a shorter time to substrate cleavage in these tumour cells, which was associated with a higher rate of apoptotic activity, suggesting these patients were more likely to respond to adjuvant treatment.
In vivo toxicity study was performed to achieve the maximum tolerated dose for the combination of TMZ and Birinapant in rats, followed by haematological and biochemistry profiles of the treatment regimes. Responses in vivo were analysed in an orthotopic xenograft GBM model. The toxicity study, using the same treatment schedule, showed a higher degree of tolerance of combination treatments in immunocompromised than immunocompetent rats. The acute inflammatory response, as confirmed from significant level of neutrophils as well as high level of glutamate dehydrogenase and lipase, suggesting liver and pancreatic failure in immunocompetent rats. In an orthotopic mouse GBM model, application of patient derived GBM cells, which are aggressive and poorly differentiated, revealed resection of the tumour did not confer survival advantage when compared to the non-resection group. Furthermore, a Type C patient-derived cell line that was TMZ-insensitive in vitro and showed a strong sensitivity to TMZ and TMZ plus Birinapant treatments. Our results demonstrate remarkable differences in responses of patient-derived GBM cells to Birinapant single and combination treatments and suggest that therapeutic responses in vivo may be greatly affected by the tumour microenvironment. Thus, in preclinical testing of new therapeutics, a careful experimental design to include both in vitro and in vivo study are warranted for a well-balanced experimental interpretation.
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Zakaria Z. Preclinical evaluation of apoptosis resensitisation with Inhibitor-of-apoptosis-protein (IAP) antagonist in glioblastoma cell lines and in an orthotopic mouse GBM model [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2016.