Tyrosine kinase inhibitors (TKIs) are currently the first therapy option for chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients. However, many patients affected by CML and AML may develop resistance to TKIs or may not recover under this treatment regime. New potential and more effective treatments are recently emerging. Heat shock protein inhibitors (HSPIs) and the proteasome inhibitor Bortezomib are drugs which have been yet to be successfully tested on leukemic patients, despite being successful on other malignancies such as multiple myeloma (MM). The combination between HSPIs and Bortezomib could potentially be successful in killing leukemic cells, by enhancing their respective molecular mechanisms. Indeed, HSPIs would bind to HSP72 avoiding the protein to exert its ligase function to the proteasome, whilst Bortezomib could stop the ubiquitinated proteins to enter the proteasome and ultimately inducing apoptosis. To test the effects of such combination, cell viability was measured via MTS assay, apoptosis levels were tested through Annexin V\PI assays. Involvement of HSP72 and pro-survival protein Bcl-2 were measured via flow-cytometry. The cells were administered with HSPIs and Bortezomib first as single agents for 24 hours, to establish working minimal concentration. Also, the drugs were tested for a shorter time, to understand when the drugs start to be effective. It emerged that one hour is sufficient for the drugs to give an initial effect in terms of cell viability and apoptosis. Following, combination experiments of HSPIs and Bortezomib were performed; the first drug was administered for one hour, the second following one hour and the cells were incubated for 24 hours. This was repeated alternatively for both type of drugs on the different cell lines. MTS and Annexin V\PI showed that there is not a synergistic effect between drugs, but instead there is antagonism. No necrosis was found at any level of the study. The cells were then probed for HSP72 and Bcl-2, to investigate their involvement in apoptosis mechanisms. Following 6 hours of combined and single agent treatment, both type of drugs inhibit HSP72 but failed to reduce the expression of Bcl-2, particularly on AML cells. It is thus proposed that CML and AML cells may die by apoptosis following a short time of treatment with HSPIs and Bortezomib by an extrinsic pathway of apoptosis, independent from Bcl-2 involvement and from mitochondrial pathway of apoptosis. This study may be the first to indicate a potential use of HSPIs and Bortezomib on CML and AML patients for a short time of treatment, although not in combination. Future studies are needed to further investigate the mechanisms of action of these drugs, aiming to potentially give CML and AML patients another successful therapy option to overcome resistance to canonic chemotherapy.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:768125 |
| Date | January 2019 |
| Creators | Marsico, Paolo |
| Contributors | Williams, John ; Ireland, Elyse |
| Publisher | University of Chester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10034/621798 |
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