Document Type

Theses, Ph.D

Rights

Available under a Creative Commons Attribution Non-Commercial Share Alike 4.0 International Licence

Publication Details

Thesis submitted to the Technological University Dublin for the award of Doctor of Philosophy, November 2017.

Abstract

A new class of Cu(II) phenanthroline-phenazine complexes (developed in Dublin City University) have recently been reported to have the highest DNA binding constants of any copper therapeutic complex and at a similar level to Actinomycin. These new complexes show great clinical potential with their high activity against a range of in-vitro mammalian cell lines including those with cisplatin resistance however the underlying biological mechanism of their action has yet to be explored in-depth. The biological data generated in this project has examined the activity of the Cu(II) phenanthroline-phenazine complexes in selected mammalian cells lines in-vitro and demonstrated that the Cu(II) complexes are highly active against cisplatin resistant (SKOV-3 and A2780/ Cis) cells. The use of γH2AX a DNA DSB marker demonstrated a high number of foci in cisplatin resistant (SKOV-3) cells compared to the cisplatin sensitive (MCF-7) cells. The strong detection of mitochondrial outer membrane potential (MoMP) decay in resistant (SKOV-3 and A2780/ Cis) cells after exposure to the Cu(II) phenanthroline-phenazine complexes was complemented by the potential disruption of mitochondrial protein homeostasis, with resultant mitochondrial toxicity and apoptosis induction detected by gene expression studies. The work was then complemented by the untargeted whole cell quantitative LFQ proteomics method. Proteomics of both resistant (SKOV-3) and sensitive (MCF-7) cell lines demonstrated increased production of metal sequestration by metallothioneins (Metallothionein-2, -1X) and, heat shock (HSP70) and ribosomal proteins (40S and others) however, metabolic proteins (glycolysis/ gluconeogenesis, fatty acid synthesis and purine metabolism related) tended to dominate the repertoire of the resistant cells. This evidence suggests the response of the cell to the Cu(II) phenanthroline-phenazine complexes is associated with its energy status. Parallel in-vivo studies using the G. mellonella model was carried out with the Cu(II) phenanthroline-phenazine complexes to examine the in-vivo biological mechanistic response to the Cu(II) complexes. The Cu(II) complexes presented a superior toxicity profile when compared to cisplatin and through high-resolution LFQ proteomics it was determined that the in-vivo response was characterised by metabolic enzymes (glycolysis/ gluconeogenesis and purine metabolism related) and detoxification processes (GST). Both in-vitro and in-vivo data generated in this project demonstrated that the mitochondria and associated metabolic factors in addition to the DNA interactions inducing apoptosis play a vital role in the mechanism of action of the Cu(II) complexes. However, this is not withstanding the fact that this work also provided clues to other associated mechanisms of action which warrants further investigation. This work demonstrated for the first time the molecular mode of action of this class of novel Cu(II) phenanthroline-phenazine complexes in both in-vitro and in-vivo biological models. The strong activity of these Cu(II) complexes against cisplatin resistant cells has the potential to offer a novel and alternative therapeutic option to the challenge of cisplatin refractory cancers, which is a high priority in clinical oncology.

DOI

https://doi.org/10.21427/j6jb-dq08

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