I hypothesise that cancer cells with high aldehyde dehydrogenase (ALDHhigh) activity present a new therapeutic target and will be selectively sensitive to 5-nitrofuran pro-drugs. Cancers are heterogeneous and contain subpopulations of ALDHhigh cells with tumour initiating potential. ALDH enzymes metabolize toxic aldehydes, and are highly expressed in somatic and cancer stem cells (CSCs), although their function in CSCs is not fully understood. In a small molecule screen coupled with target ID, Zhou et al. (2012) recently discovered that clinically active 5-nitrofurans (5-NFNs) are substrates of ALDH2. 5-NFNs are a class of pro-drug widely used to treat bacterial and parasitic infections, where their relative specificity is driven by nitroreductases, but little is known about the enzymes that bio-activate 5-NFNs in humans. Recent clinical cancer research has found that the 5-NFN, nifurtimox, has anti-cancer properties and it is currently in Phase 2 clinical trials for neuroblastoma and medulloblastoma (ClinicalTrials.gov Identifier: NCT00601003), however the mechanism underlying this anti-cancer activity is unknown. In melanoma and other cancers, ALDH1A1 and ALDH1A3 are highly expressed in CSCs. I demonstrate the anti-cancer activity of 5-NFNs in cancer cell lines, where they express high sensitivity to 5-NFNs in cell viability assays (A375 melanoma cells EC50 = 867nM). To test if ALDH1 enzymes are substrates of 5-NFNs, I performed in vitro activity assays by monitoring NADH production (λ = 340nm). I found that the clinically available 5-NFNs, nifuroxazide and nifurtimox, in addition to our own newly synthesised 5-NFNs, are competitive substrates for human ALDH1A3 activity in vitro (P < 0.05). Notably, nifuroxazide is not a substrate for ALDH2, suggesting that nifuroxazide may show selectivity toward ALDH1. Enzymatic assays with purified human ALDH2, demonstrate that ALDH2 requires NAD+ for bio-activation of 5-NFNs. Consistent with these assays, I found that 5-NFNs are competitive substrates for ALDH activity in melanoma cells by Aldefluor™, with 5-NFNs displaying a prolonged competitive inhibition of ALDH activity compared with the known inhibitor, DEAB. Importantly, no-nitro control compounds show no activity toward ALDH enzymes in vitro or in culture. Kinetic living-cell imaging (IncuCyte ZOOM®) reveals that a subpopulation of ALDH1A3 siRNA transfected A375 cells are protected from 5-NFN toxicity (P > 0.05) and cell death (DRAQ7™: P < 0.0001), demonstrating a functional role for ALDH1A3 in mediating 5-NFN activity in cancer cells. In contrast, A375 cells overexpressing ALDH1A3 by cDNA transient transfection were hypersensitive to 5-NFNs (P < 0.001), determined by Muse™ cell viability. Computational docking studies reveal that 5-NFNs have the potential to fit within the interior of the ALDH enzymatic cavity and interact with the catalytic cysteine, thereby offering a potential mechanism for 5-NFN bio-activation. Finally, in collaboration, we show a unique interaction between 5-NFNs and ALDH using mass spectrometry and have initiated protein crystallography trials. My work demonstrates a novel and biologically relevant 5-NFN-ALDH interaction in cancer cells. I propose 5-NFNs have the potential to target ALDHhigh CSCs within a tumour and advance the repurposing of clinical 5-NFN pro-drug antibiotics as anti-cancer therapeutics.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:736024 |
| Date | January 2017 |
| Creators | Crispin, Richard Kean |
| Contributors | Patton, Elizabeth ; Unciti-Broceta, Asier |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/28716 |
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