Evaluation of two xenobiotic reductases from Pseudomonas putida for their suitability for magnetic nanoparticle‐directed enzyme prodrug therapy as a novel approach to cancer treatment

Abstract

Traditional cancer treatment methods, including surgery, radiotherapy, and chemotherapy, have progressed over recent years; however, there are still many limitations associated with these treatments such as the lack of selectivity between healthy and cancerous cells (Braun & Seymour, 2011; Brown & Giaccia, 1998). One approach being investigated to improve the selectivity of chemotherapy

The YfkO Nitroreductase from Bacillus Licheniformis on Gold-Coated Superparamagnetic Nanoparticles: Towards a Novel Directed Enzyme Prodrug Therapy Approach

“…Whilst this appears to be contrary to what might be expected, it could be attributed to a well-documented effect, known as the Hormetic effect [ 40 ]. The Hormetic effect is evident from the characteristic inverted- U-shaped response on the graph [ 40 ], and it is a phenomenon that we have come to expect when testing CB1954’s cell killing potency in SK-OV-3 cells [ 32 , 33 , 34 ].…”

“…Cell viability assays carried out by Vass et al on the SK-OV-3 cell line appeared to show superior results in terms of the cell viability after treatment compared to the data reported in this study [ 44 ]. An explanation for this difference in reported cell viability is that, in their work, they incubated their cells with the additional cofactor (NAD(P)H) whereas in this dataset only the intracellular cofactor is used, with no additional cofactor being added as this gives a better indication of how the treatment will perform in vivo [ 32 , 33 , 34 ]. The fact that a killing of the cells was observed indicates that the treatment was successfully up taken into the SK-OV-3 cells, where it could access the intracellular cofactor to produce toxic products and induce cell death.…”

“…CB1954 kinetic experiments were all carried out in a 96-well microtiter plate (Corning, USA) using a Thermoscientific Varioskan 96-well plate microplate reader using the method that we have previously described [ 32 , 33 , 34 ]. Product formation was measured at 420 nm over time to determine the -Menten kinetic parameters of the CB1954 prodrug with either NfnB-Cys or YfkO-Cys.…”

“…All HPLC experiments were carried out on a UHPLC machine (Dionex Ultimate 3000 HPLC system, ThermoScientific, Waltham, MA, USA) using a C18 column for analysis (Waters Spherisorb ® 5 µm ODS2 4.6 mm × 250 mm C18 column, Wilmslow, UK) following the method previously described [ 31 , 32 ]. HPLC runs were performed with the instrument set to the following parameters: 50 µL injection volume, a fixed column oven temperate of 25 °C, a run length of 45 min and the UV wavelength for detection was 420 nm [ 31 , 32 , 34 ]. Owing to the light sensitive nature of some of the reagents, samples that were designated for HPLC were prepared in 15 mL amber falcon tubes as follows: NADH (120 µL, 10 mM) nitroreductase (116 µg/mL), CB1954 (20 µL, 50 mM) then made to a final volume of 1080 µL using PB (50 mM, pH 7.2).…”

“…Cell viability assays were performed as previously described [ 32 , 33 , 34 ]. SK-OV-3 (ECACC 91091004) ovarian cancer cells (Sigma–Aldrich, Gillingham, UK) were seeded into a 96-well plate (corning, Corning, NY, USA) at a density of 1 × 10 3 cells per well, in 100 µL Dulbecco’s Modified Eagles Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 1% penicillin/streptomycin and 1% l -glutamine, and were left to attach to the plate in a 5% CO 2 incubator at 37 °C overnight.…”