The contribution of reactive nitrogen species to the cytotoxicity of nitric oxide generating therapy

Nitric oxide (NO) has become increasingly recognized throughout the past decade for its role in many patho-physiologies. NO delivery has been investigated as a potential therapeutic strategy against solid tumours, using both NO donor drugs and gene therapy strategies. This project aims to study the importance of oxygen concentration and the possible role of key reaction products involved in NO induced cytotoxicity. Utilising the NO donor drug, DETA/NO the effects of oxygen tension on the cytotoxicity was investigated in vitro. The find ings presented in this study indicate that the DETA/NO mediated cytotoxic effects in all the tumour cell lines tested was significantly more toxic under severe hypoxia. This effect was most evident at drug concentrations greater than 10 ~M. Both the intrinsic and extrinsic apoptotic pathways were activated fo llowing DETA/NO treatment, and cleavage of apoptotic proteins was enhanced under hypoxia. DETA/NO treatment under hypoxia significantly destabilized hypoxia inducible factor (HIF-1a) and resulted in the accumulation of p53 protein. Investigations into the contribution of reactive nitrogen species to NO' cytotoxicity revealed that neither peroxynitrite (ONOO-) nor Nitroxyl (HNO) was generated. Fluorometric analysis in the presence of dinitrogen trioxide (N,O,) scavengers suggests for the first time that N,O, may be responsible for the cytotoxicity with DETA/NO. Compelling evidence is provided to suggest that S-nitrosylation is the key molecular mechanism involved in NO signalling. This is the fi rst study that shows significant Snitrosothiol formation in cancer cells when exposed to high levels of NO irrespective of oxygen concentration. Further investigations revealed that both nuclear p53, and GAPDH proteins are targets for S-nitrosylation following treatment with DETA/NO, and may possibly play a key role in DETA/NO-mediated apoptosis and cytotoxicity.EThOS - Electronic Theses Online ServiceGBUnited Kingdo