The current outlook for patients suffering malignant glioma is poor, with suboptimal 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 realtime 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.
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 facto
|Date of Award||Jun 2011|
|Supervisor||Geoff Pilkington (Supervisor) & Arthur Butt (Supervisor)|