RNASeq and patient outcome data was retrieved from the Broad GDAC Firehose via the Firebrowse data portal for the Cancer Genome Atlas (TCGA) liver hepatocellular carcinoma (LIHC) cohort (https://gdac.broadinstitute.org/); and use of the TCGA dataset was approved by the NIH as part of our project entitled #21012: “Investigating Novel Genes in HCC”. RNASeq data was normalised and differential expression of genes of interest between tumour and normal samples was evaluated using the R/Bioconductor package ‘DESeq2’, with significant differential expression defined by an adjusted p value < 0.05. Based on gene expression in tumour tissue, patients were separated into cohorts with ‘High’, ‘Medium’, or ‘Low’ expression of each gene of interest. Using patient outcome data, patient median survival was calculated and plotted using the R/Survival package, and significant difference in survival between cohorts was calculated using the log-rank test.
Huh7 HCC cells were cultured in DMEM media supplemented with 10% heat-inactivated FBS and 1% (v/v) Penicillin–Streptomycin (Gibco). A52 HCC cells were used for a murine model of HCC as published by us16 and cultured in DMEM supplemented with 20% heat-inactivated FBS, 20 μg/L human epidermal growth factor (hEGF; Sigma Aldrich), 0.01 g/L insulin (Gibco), 0.01 g/L hydrocortisone, 1 mM phenobarbital, and 1% (v/v) Penicillin–Streptomycin. Cells were maintained at 37 °C in 5% CO2.
Cells and tumour tissue were lysed in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM sodium orthovanadate, 50 mM NaFl, 1 mM sodium molybdate, 40 mM β-glycero-phosphate, 1 mM PMSF and 1/100 protease inhibitor cocktail) and protein concentration of lysates was determined using DC Protein Assay Kit (Bio-Rad). A total of 25 μg of protein was subjected to gel electrophoresis on a 10% SDS-PAGE gel and transferred to a PVDF membrane for one hour at 100 V. Membranes were washed in TBST (Tris-buffered saline with 0.2% Tween 20) and blocked with 5% skim-milk/TBST. Membranes were incubated at 4 °C overnight with the following primary antibodies: G6PD (12263, Cell Signaling), PSAT1 (ab96136, Abcam), PHGDH (ab13428, Abcam), transketolase (8616, Cell Signalling), transaldolase (PA5-27614, ThermoFisher) and β-actin (A2228, Sigma Aldrich), Membranes were washed in TBST and incubated with their respective HRP-conjugated secondary antibodies (Sigma) in 5% skim-milk/TBST, washed with TBST and visualised using SuperSignal West Pico PLUS Chemiluminescent Substrate kit (34577, ThermoFisher). Original blot images can be found in Supplementary File 1. Densitometry analysis was performed using ImageJ and normalised to β-actin expression to determine the adjusted density value for each protein.
In vitro effect of fructose, NCT-503, and Physcion on HCC cell proliferation
Huh7 and A52 cells were seeded at 1.0 × 104 cells and 2.0 × 103 cells per well, respectively, in 96-well plates. After 24-h, the media was replaced with fresh media containing either standard media, glucose-free DMEM media (Gibco) supplemented with 10% heat-inactivated dialysed FBS, and 5 mM glucose or 5 mM fructose. For drug experiments, the media was supplemented with 10, 25, or 50 μM of NCT-503 (HY-101966, MedChemExpress) and/or Physcion (M4323, Abmole). Amino acid media supplement was a 1/100 dilution of MEM non-essential amino acid solution (M7145, Sigma Aldrich). All treatment groups were conducted in quadruplicate, and cell proliferation was determined after 24-h and 48-h with a BrdU Colorimetric assay (Roche), according to the manufacturer’s protocol.
In vitro effect of NCT-503 and Physcion on HCC tumoursphere formation
Huh7 and A52 cells were cultured in sphere assay media consisting of 2% B27 Supplement (Gibco), 20 ng/mL hEGF, 10 ng/mL basic fibroblast growth factor (bFGF; Gibco), 2 μg/mL heparin (Sigma Aldrich), 5 μg/mL insulin, 0.5 μg/mL hydrocortisone, and 1% (v/v) Penicillin–Streptomycin. Huh7 and A52 cells were seeded at 3.0 × 104 cells per well in 6-well low attachment plates (Sigma Aldrich) and incubated at 37 °C in 5% CO2 for seven days. Treatment groups included sphere formation in media containing either 5 mM glucose or fructose alone, or glucose or fructose-supplemented media containing NCT-503 and/or Physcion. After 7 days, tumourspheres were counted.
Huh7 and A52 cells were cultured in normal media (negative control), glucose media, or fructose media, as described above, for 48 h. For a positive control, fresh media containing 1 mM H2O2 was supplied to A52 and Huh7 cells 2 h prior to experimentation to induce apoptosis. For apoptotic cell analysis, cells were harvested with Accutase and stained with FITC-Annexin V (640906, BioLegend) and 7-AAD (7-aminoactinomycin D; 420404, BioLegend). Flow cytometry was performed using a BD LSRFortessa and BD FACSDIVA software (Version 6.1.3, BD Sciences). The FITC channel and PerCP-Cy5-5 channel were used for Annexin V and 7-AAD emission wavelengths, respectively. A total of 10,000 events were recorded for each group and FlowJo version 10.6.2 was used for data analysis and graphing. Quadrant placement (FITC-A versus PerCP-Cy5-5) was based on unstained and single stained cells from the negative and positive control treatment groups, and represent Q1 (non-specific cell death), Q2 (late stage apoptosis), Q3 (early stage apoptosis) and Q4 (viable cells).
In vivo effect of NCT-503 and Physcion in glucose and fructose-fed HCC tumours
All animal experimental protocols were approved by the James Cook University Animal Welfare and Ethics committee (A2238). All experiments were performed in accordance with relevant guidelines and regulations. Male C57BL/6 mice or male BALB/cFox1nu mice aged 6–12 weeks were given ad libitum access to food and water. Huh7 and A52 HCC tumour growth was evaluated in male BALB/cFox1nu and male C57BL/6 mice, respectively. Mice were fed isocaloric diets of a normal chow (AIN93G, Specialty Feeds; protein 19.4%, fats 7%, carbohydrate 56.8%, crude fibre 7% and acid detergent fibre 7%) or a 60% fructose chow (SF03-018, Specialty Feeds; where fructose replaced sucrose, dextrinised starch and starch) for three weeks prior to HCC cells (2.5 × 106 cells in 200 μL PBS/ECM gel; Sigma Aldrich) being bilaterally and subcutaneously injected into the rear flanks of the mice (n = 6–12 each group). For drug experiments, tumours were allowed to grow to a half-maximum size of 500 mm3 to mimic tumours observed in the clinic prior to drug treatment. Mice were randomly allocated into the following treatment groups: (i) placebo (vehicle only), (ii) 40 mg/kg NCT-503 (dissolved in 60% of 30% 2-hydroxypropyl-β-cyclodextrin, 30% PEG-300, and 10% ethanol), (iii) 20 mg/kg Physcion (dissolved in 10% DMSO and 90% saline), or (iv) 40 mg/kg NCT-503 and 20 mg/kg Physcion. Each drug or placebo was administered daily as a 200 μL intraperitoneal injection for a 14-day treatment period or until the tumour reached a maximum volume of 1000 mm3. Tumour volume was measured daily with a digital caliper and calculated using the following formula: V = (L × W2/2), where V is volume, L is length, and W is width. To determine differences in tumour growth non-liner regression was performed with the Prism program. At experiment end the tumours were harvested, weighed and snap-frozen in liquid nitrogen or fixed in 4% paraformaldehyde for histology, stained with haematoxylin and eosin, and scanned with the Aperio ImageScope. The haematoxylin ratio was determined with ImageJ.
Data and statistical analysis
All data are presented as mean ± standard error. Data from different experimental groups were compared using one-way and two-way ANOVA and Tukey’s multiple comparison tests. For densitometry analysis, the 2 treatment groups were compared using the Mann–Whitney t test.
Statistical significance was defined as p < 0.05.