Nox1 inhibitor - GKT310771
GKT310771 is a potent, selective and specific NOX1 inhibitor (Ki =65 ± 30 nM) with an affinity similar to diphenyliodonium (DPI; Ki =70±5 nM) that is an irreversible and unspecific flavoprotein inhibitor (Genkyotex, Saint-Julien-en-Genevois, France). GKT310771 is 65-fold less effective against NOX4 (Ki = 4290 ± 437 nM) and does not inactivate NOX2, NOX3 and NOX5. It is specific for NADPH oxidases over other flavoenzymes and exhibits no ROS scavenging or antioxidant activity according to the absence of affinity for xanthine oxidase and DPPH (Ki>100μM). An extensive in vitro off-target pharmacological profiling of GKT310771 against various proteins including ROS-producing and redox-sensitive enzymes, as well as recognized drug targets (GPCRs, kinases, ion channels and others) failed to reveal significant inhibition of any tested candidate when used at 10 μM, thereby demonstrating high specificity of GKT310771 for NOX enzymes.
HCC cell culture
The human Hep3B and murine Hepa1-6 cell line were purchased from American Type Culture Collection (ATCC, Molsheim, France). The human Huh-7 cell line was provided by dr. Francesca Fornari (27). All cell culturing agents and materials were purchased from Life Technologies, Ghent, Belgium; unless stated differently. Cell lines were cultured according to the ATCC guidelines, incubated at 37 °C under 5% CO2 and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Hep3B and Hepa1-6) or Roswell Park Memorial Institute-1640 (RPMI-1640; Huh-7), supplemented with 10% Fetal Bovine Serum (FBS) and 1% Antibiotic Antimycotic Solution (AA; containing 10’000 units penicillin, 10 mg streptomycin and 25μg amphotericin B per mL). For subculturing, cells were dissociated using 0.05% trypsin, stained with trypan blue, and counted using the LUNA-FL™ Dual Fluorescence Cell Counter (Logos Biosystems, Annandale, VA, USA).
Cytotoxicity assays
To determine the concentration-dependent cytotoxicity of the specific NOX1 inhibitor GKT771 (Genkyotex, Saint-Julien-en-Genevois, France), the cells were seeded at a density of 15×103 cells per well in 96-well culture plates, and exposed to various GKT771 concentrations (1.25 – 100 µM in 1% dimethylsulfoxide (DMSO)-enriched culture medium), or equal volumes of solvent, DMSO-free medium or positive controls (2 µM staurosporin for MTT and 2% Triton-X for LDH) for 24-48h.
The 3-(4,5-dimethylthiazol-2-yl)-2,5-difenyltetrazolium bromide (MTT) assay (Roche Diagnostics, Anderlecht, Belgium) was used according to the manufacturer’s protocol to determine the mitochondrial metabolic activity, a measure for cellular viability. Briefly, cells were incubated with 0.5 mg/ml MTT for 2h at 37°C. Mitochondrial dehydrogenases degrade MTT into insoluble formazan crystals and the absorbance of these DMSO-solved crystals was measured at 570 nm against a background control of 630 nm with a spectrophotometer (Multiskan Ascent). The results are shown as percentages of the solvent-treated control group.
The supernatant of the treated cells was used to measure the lactate dehydrogenase (LDH) activity according to the manufacturer’s protocol (Biovision, Milpitas, California, USA). The cytosolic enzyme LDH is released into the culture medium following cytotoxic damage-mediated plasma membrane disruption. LDH was quantified via a coupled enzymatic reaction in which LDH catalyses lactate to pyruvate through NAD+ reduction. The released NADH is used by diaphorase to reduce a tetrazolium salt into a red formazan product. The absorbance of the latter was measured at 490 nm against a background control of 630 nm.
The luminescent CaspGlo® 3/7 (Promega, Leiden, The Netherlands) was used to measure caspase-3 and -7 activities of the different adherent cell cultures. Upon cell lysis, the proluminescent caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, is cleaved by caspase-3/7. This releases aminoluciferin, which in turn is used by luciferase for the production of light, allowing quantification of the apoptotic state of the cell cultures. All cells were incubated in the dark for 2h with the CaspGlo® 3/7 reagent prior to detection with a luminometer (Fluostar, BMG Labtech, De Meern, The Netherlands).
Mouse BMDM and human monocyte cell line THP1
Murine bone marrow-derived macrophage single cell isolates were obtained from mice by flushing the femurs and tibiae with ice-cold phosphate-buffered saline (PBS). After centrifugation (7 min; 1200 rpm; 4˚C), the pellet was resuspended in pre-heated (37°C) DMEM supplemented with 20 ng/ml murine macrophage colony-stimulating factor (M-CSF, PeproTech, London, UK), 50 µg/ml Gentamycin (from 3 mg/ml stock), 10% FBS and 1% AA. Cells were seeded in two 10 cm petri dishes per mouse, with each containing approximately 20x106 primary bone marrow-derived macrophages (BMDMs) after 7 days of culturing. Medium was refreshed every other day and cells were collected using enzyme-free dissociation buffer. For the human THP1 monocyte cell line, cells were cultured in suspension in RPMI-1640 supplemented with 10% FBS. Prior to treatment with GKT771, cells were seeded into well-plates and stimulated with 12-O-tetradecanoylphorbol-l3-acetate (PMA, 10 ng/ml) to allow differentiation into macrophages. Concentration-dependent cytotoxicity of GKT771 with and without lipopolysaccharide (LPS)-stimulation (1µg/ml; Sigma-Aldrich) on mouse and human macrophages was determined via the MTT and LDH assay, as described above.
Mice
All animal experiments were reviewed and approved by the Animal Ethics Committee of the Faculty for Medicine and Health Sciences, University Ghent (ECD18/50). Five-week-old male SV129 wild-type mice, purchased from Janvier Labs (Le Genest-Saint-Isle, France), were housed at room temperature and constant humidity in a 12-hour controlled dark/light cycle at the animal facility of the Faculty of Medicine and Health Sciences, Ghent University, Belgium. Mice received standard chow (Pavan Service-Carfil, Oud-Turnhout, Belgium) and water ad libitum before and during the experiment. The welfare of all animals was evaluated daily and the mice were weighted weekly, during the entire duration of the experiment.
Preparation of vehicle and dose formulation
Vehicle consisted of deionized water containing 1.2w% methylcellulose and 0.1w% Polysorbate 80 (Tween 80), which was stirred overnight at 4°C to allow complete solubilisation. GKT771 was dissolved in vehicle solution. Both solutions were stored at 4°C, protected from light and stirred for at least 10-15 minutes before oral administration (dosage of 30 mg/kg).
Preventive study
In order to induce HCC, mice received weekly intraperitoneal (IP) injections of saline or diethylnitrosamine (DEN; 35 mg/kg, Sigma-Aldrich, Diegem, Belgium) for 25 weeks. At week 15, mice were randomised into 4 treatment groups and treated for 15 weeks: vehicle-treated healthy saline controls (n=6), GKT771-treated healthy saline controls (n=4), vehicle-treated DEN-induced HCC mice (n=7) and GKT771-treated DEN-induced HCC mice (n=10). Mice were treated daily via oral gavage. At week 30 of the experiment, mice were sacrificed (Figure 1A).
Therapeutic study
Two different treatment regimens were applied in the therapeutic setting of the experimental set-up. As in the preventive setting, mice received weekly IP injections of saline or DEN. This was maintained for 25 weeks in the early treatment regimen and 20 weeks in the delayed treatment regimen. Since HCC nodules are macroscopically visible as soon as 20 weeks of DEN (28), an early treatment regimen was started in which mice were planned to be treated with vehicle (n=11) or GKT771 (n=9) twice daily via oral gavage for a total of 10 weeks. However, due to ethical reasons, early euthanasia was performed after 9 weeks. A second treatment regimen was set up in which mice were kept in a resting period of 2 weeks after the final DEN injection at week 20. During this delayed treatment regimen, mice were treated daily with vehicle (n=5) or GKT771 (n=6) for 5 weeks via oral gavage (Figure 1B).
Sample collection
At the day of sacrifice, mice were weighted, and anesthetized by intraperitoneally injecting a ketamine (100 mg/kg) – xylazine (10 mg/kg) solution for blood sampling. The mice were euthanized via cervical dislocation prior to dissection. Both spleen and liver were isolated and weighed, after which the number of nodules was macroscopically evaluated. The lesions of the mice from the preventive study and of the treatment regimen 2 of the therapeutic study were manually separated from the non-tumorous liver tissue. Liver tissue without tumors, the tumors and the total livers were divided into different sections for further analyses. Sections for RNA analysis were collected in RNA-later, snap-frozen using liquid nitrogen and stored at -80°C until further analysis. Tissue sections for histological examination were collected in cassettes and fixed in a 4% phosphate-buffered formaldehyde solution, and embedded in paraffin.
Total RNA extraction
For the quantification of specific gene transcript levels, needle homogenisation was performed on all tissue samples prior to lysis with RLT lysis buffer supplemented with β-mercaptoethanol (Biorad, Temse, Belgium). Total RNA was extracted from all samples using the Aurum™ Total RNA Mini Kit according to the manufacturer’s Spin Format Guidelines (Biorad, Temse, Belgium). The purity and quantity of total RNA was assessed using spectrophotometry (Nanodrop, Thermo Scientific, Wilmington, USA). Purity was determined using the ratio of absorbance at 260 and 280 nm (only ratios between 1.8 and 2.0 were accepted).
Quantitative real-time polymerase chain reaction
One microgram of total RNA was converted to single strand cDNA by reverse transcription (Sensifast cDNA synthesis kit, Bioline Reagents Ltd, Kampenhout, Belgium), following the cDNA Bio thermocycler program (10’ 25°C, 15’ 42°C, 5’ 85°C). The cDNA was diluted 1/10 prior to real-time quantification via reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) using SYBR Green (NO-ROX, Sensimix, Bioline Reagents Ltd., Waddinxveen, The Netherlands) according to the manufacturer’s guidelines. All reactions were performed in duplicate. All used primer sequences (Biolegio, Nijmegen, The Nederlands) are listed in table 1. A real-time cycling program (initial denaturation: 10’ 95°C; 40 PCR cycles: denaturation 10” 95°C and annealing/extension/fluorescence reading 60” 60°C) was run on a Lightcycler® 480 II (Roche, Machelen, Belgium) and melting curve analysis was performed to assess primer specificity. The threshold cycle (Ct) values were further analysed using the 2-ΔΔCT method. In short, the average of the in duplo Ct values of the household and target genes were calculated. The geometric mean of these values of the stable household genes (determined via GeNorm) were calculated and subtracted from the in duplo averages of the target genes (= ΔCp). Next, the mean of the ΔCp of the control samples was subtracted from the ΔCp of each individual sample (= ΔΔCp) and the power was calculated (=2-ΔΔCT). All expression analysis graphs display these powers, and statistical analysis was performed on the normalized log10 transformed data of these powers. Statistical analyses were performed as described below.
Luminex
Snap-frozen liver tissue was defrosted in 1 mg/mL protease inhibitor cocktail (cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail, 04693 159 001, Roche, Sigma-Aldrich, Overijse, Belgium), 1 v% phosphatase inhibitor cocktail 2 (P5726, Sigma-Aldrich, Overijse, Belgium) and 1 v% phosphatase inhibitor cocktail 3 (P0044, Sigma-Aldrich, Overijse, Belgium) in PBS, lysed by sonication and centrifuged for 15 min by 15,000 rpm at 4 °C. Supernatant was stored at −80 °C until further analysis. Total protein concentrations were measured by BCA protein assay following manufacturer's guidelines (DC protein assay, 500-0116, Bio-Rad, Temse, Belgium). Protein levels of TNF-α, MCP-1/CCL2 and IL6 were determined by using Bio-plex Pro Reagent Kit and Luminex microbeads (Biorad, Temse, Belgium) according to the manufacturer's guidelines.
ROS assay
Hepatic tissue lysates were prepared by defrosting snap-frozen liver tissue in PBS prior to sonication on ice. The lysates were centrifuged for 5 min by 10,000 g at 4 °C. The supernatant was used for the quantification of ROS levels, which was performed using a commercial DCF ROS/RNS Assay Kit (Ab238535, Abcam, Cambridge, United Kingdom,) according to the manufacturer’s instructions.
Histological analysis
After deparaffinization and rehydration of the tissue samples, sections of 5 μm of paraffin-embedded liver tissue were stained with Sirius Red (Sigma-Aldrich) for histopathological examination according to the Metavir scoring system. This system allows to assess the extent of fibrosis in our liver samples, which was visualised using the Cell^D software (Olympus).
Statistical analysis
Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, California, USA). Normality was tested using the D’Agostino & Pearson omnibus test. Outliers were identified with the ROUT method and were removed for further analysis. The maximum desired false discovery rate was set to 1%. Gaussian distributed data of two groups were compared via t-tests and multiple groups were compared by one-way analysis of variance (ANOVA) test and corrected with the Holm-Sidak test. Non-normally distributed data were either transformed to Gaussian distribution via LOG10-transformations or analysed via Kruskal Wallis tests for multiple comparison. Data are presented as the fold change relative to expression in controls as mean ± SD, unless stated differently in the figure legends. P-values are reported two-sided and considered significant when less than 0.05 (*p<0.05, ** p<0.01, *** p <0.001, **** p<0.0001).