Hayden, Rachel Elizabeth (2010)
Ph.D. thesis, University of Birmingham.
Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in the western world, and continues to have very low cure rates. The bulk of the tumour cells are non-cycling peripheral cells that accumulate from a minority of dividing cells within proliferation centres (PCs), located the lymph nodes (LN) and spleen. The PCs are rich in activated T helper (TH) cells that express CD40L and secrete IL-4. Like normal B cells, CLL cells interact with TH via their expression of CD40 and receive signals from IL4. This stimulation provides the signal for B cells to divide, as well as protecting them from apoptotic stimuli. Consequently, many current therapies are unable to sufficiently target the cells in the PC, resulting in therapy resistance, residual disease (RD) and relapse. In addition, most therapies have associated toxicities, restricting their use to younger, fitter patients. Thus, there is an urgent need for new therapies with low toxicity, especially for older patients. New drug discovery is time consuming and expensive. Redeployment of existing drugs is one way to potentially combat this issue. The lipid lowering drug Bezafibrate (BEZ) and the sex steroid, medroxyprogesterone acetate (MPA) have been shown to have anti-leukaemic properties in other settings. In this study the in vitro efficacy of BEZ and MPA on resting CLL cells (representing resting cells in the periphery) and when stimulated to proliferate by CD40L (representing cells in the PCs) have been investigated. Both BEZ and MPA exerted pro-apoptotic actions against resting CLL cells and reduced CD40L stimulated proliferation. These actions were increased when both agents were combined and demonstrated selectivity against CLL cells compared to normal peripheral blood mononuclear cells. Combined BEZ+MPA, was as effective as the commonly used chemotherapeutic chlorambucil, with the combination of all 3 agents exerting the greatest effects. However none of the combinations induced apoptosis of CLL cells protected by CD40L. Investigations into the possible mechanisms of drug action revealed that MPA was unlikely to be working either by steroid receptors or by inhibition of the enzyme AKR1C3 and the mechanism remains unknown. BEZ alone and in the presence of MPA induced the production of prostaglandin D2 (PGD2) and exogenously applied PGD2 was found to exert apoptosis in a similar manner to BEZ. Both BEZ and MPA generated reactive oxygen species (ROS) in both culture settings and the combination was more effective than either drug alone. In the absence of CD40L, mitochondrial superoxide (MSO) was also produced but not in CD40L stimulated cells. This finding suggested that CD40L is able to prevent or protect against MSO production and, consequently, apoptosis. Attempts at overcoming this effect revealed that the plant derived compound lycorine exerted minimal effects alone but, when combined with BEZ+MPA, reinstated the induction of MSO and recapitulated BEZ+MPA induced apoptosis despite the continual presence of CD40L. In contrast, the reported ability of dasatinib to overcome CD40L mediated fluadarabine resistance was discovered to be to be unfounded.
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