Curcumin was obtained from Sigma (MO, USA). WZ35, a monocarbonyl curcumin analog, was prepared as described in prior studies . Cell Counting Kit-8 (CCK-8) and horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgG and N-acetyl-l-cysteine (NAC) were purchased from Beyotime (Haimen, China). Cell Apoptosis DAPI Detection Kit, DCFH-DA ROS detection kit, and pEGFP-hYAP 1 were obtained from Addgene (Shanghai, China). A Hippo Signaling Antibody Sampler Kit (#8579), anti-Phospho-SAPK/JNKThr183/Tyr185 (#4668), anti-Cleaved caspase 3 (#9661), anti-Bax (#2774), anti-Bcl-2 (#2870), anti-E-cadherin (#8834S), anti-GLUT1 (#73015), anti-N-cadherin (#13116S), and anti-GAPDH (#D16H11) were purchased from Cell Signaling Technology (USA).
The human HCC cell lines including HCCLM3, HepG2 and Huh7 were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were grown using Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS (Life Technologies) and penicillin/streptomycin at 37 °C in a humidified 5% CO2 incubator.
In total, 110 matched HCC tumor samples and precancerous control tissues were isolated from untreated patients undergoing surgical tumor resection at the Department of Hepatopancreatobiliary Surgery of the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, between 2010 and 2014. The tumor samples were separated into two different portions, with one portion being used for routine histopathological examinations, and the rest being snap-frozen and stored at -80 °C for future studies. HCC was pathologically confirmed in all patients, with tumors staging being determined based upon the AJCC/UICC (Union for International Cancer Control/American Joint Committee on Cancer) and WHO classification. The Board and Ethical Committee of the First Affiliated Hospital of Wenzhou Medical University approved this study（ID number：wydw2019-0446）, and all the patients provided written informed consent.
The GENE EXPRESSION OMNIBUS (GEO) database was searched using the terms "gene expression", "hepatocellular carcinoma", and "Homo sapiens". We selected the GSE14520 dataset as it met the quality control and the sample size criteria. The R software (version.4.0.3) affyPLM package was used for the regression analyses and normalization. Based on quality filtering, two healthy control microarray samples were excluded, while the remaining 41 microarrays (22 HCC samples, 19 adjacent control samples) based on the GPL571 platform (Affymetrix Human Genome U133A 2.0 Array) were retained for analysis. The RStudio Limma package was used to identify the differentially expressed genes (P < 0.05 and | logFC (Fold Change) | ≥ 1), after which probe names were converted to gene names. The heatmap.2 package was used to visualize differences in gene expression between the samples, with appropriate functions also being used to draw the boxplots, survival curves, and volcano plots in R software (version.4.0.3).We used database for annotation, visualization and integrated discovery for GO enrichment analysis.We also performed pathway enrichment analysis using pathway data obtained from the FTP service of KEGG (Kyoto Encyclopedia of Genes and Genomes). JASPAR  and USCS  were used to predicted binding sites, conservation and histone modification.
Cell proliferation assay
A CCK-8 kit was used to measure the cellular proliferation. Briefly, HCC cells were plated in 96-well plates (8x103/well) overnight, after which they were untreated or treated with curcumin (10 μg/mL), WZ35 (10 μg/mL), NAC (5 mM). For some experiments, a range of drug concentrations (0, 5, 10, 20, and 40 μg/mL) were used instead. WZ35 and curcumin were prepared using 0.03% DMSO; NAC was prepared in PBS and diluted using complete media to appropriate concentrations for use. After drug treatment, the CCK-8 kit was used to measure the viability with absorbance at 450 nm being quantified via microplate reader (Spectramax m5, Molecular Devices).
Colony formation assay
Initially, HCCLM3 cells were plated in 6-well plates (5,000/well) with curcumin (1 μg/mL), WZ35 (1 μg/mL), and/or NAC (5 mM). The plate incubated until individual colonies were formed containing approximately 50 cells. The cells were then washed in PBS, fixed for 20 minutes with methanol, stained for 15 minutes with crystal violet, washed with distilled water, and colonies were counted using the ImageJ software.
DAPI staining for cells
Apoptosis was measured by plating HCCLM3 cells in 6-well plates overnight (5×105/well). The cells were then treated using WZ35 or curcumin (both 10 μg/mL) for 18 h, after which they were washed using PBS, fixed for 15 minutes in 4% paraformaldehyde (PFA), and stained for 15 minutes with 4',6-diamidino-2-phenylindole (DAPI) (400 μL). The cells were thereafter imaged via microscope (NIKON, Japan) with 100 magnification.
Radio immunoprecipitation assay (RIPA) lysis buffer obtained from Beyotime (Haimen, China) was used to isolate the proteins from the cells or to homogenize the tumor tissues. The samples were then spun for 15 minutes at 12,000 rpm, and equal amounts of supernatant protein (10 μg/sample) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8, 10 or 12%) prior to transfer onto a poly (1,1-difluoroethylene) (PVDF) membrane (Millipore, Burlington, MA, USA). The blots were blocked for 90 minutes using 5% skim milk in TRIS hydrochloride & Tween (TBST), followed by an overnight incubation with the various primary antibodies at 4 °C. After washing three times in TBST (10 minutes/wash), the blots were incubated for 1 h with horseradish peroxidase (HRP)-linked secondary antibodies (1:2000), after which they were washed again and protein bands were detected using an electrochemiluminescence (ECL) reagent (Thermo Scientific, IL, USA).
Transmission Electron Microscopy (TEM)
Curcumin- and WZ35-induced changes in the cellular structure were visualized via TEM, with DMSO being used as a control. Initially, the treated cells were pre-fixed overnight in 2% glutaraldehyde, followed by a 2 h post-fix in 1% osmium tetroxide. Ethanol and acetone gradients were then used to dehydrate the cells, after which the cell pellets were embedded in ethanol and acetone. Ultrathin pellet sections were then stained using 2% aqueous uranyl acetate and lead citrate, after which a TEM instrument (Wenzhou Medical University, H-7500, HITACHI, Japan) was used to image the cells with 26500 magnification.
Oxidative phosphorylation and glycolysis measurements
A Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies, CA, USA) was used for oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements. Briefly, curcumin or WZ35 doses were used to treat HCCLM3 cells for 6 h, after which 2×104 cells were added to machine-specific plates. Following probe calibration, OCR was measured via sequentially injecting oligomycin (ATP synthase inhibitor; 1 µM), FCCP (uncoupler; 0.5 µM), rotenone (complex I inhibitor; 1 µM), and antimycin A (complex III inhibitor; 2 µM). ECAR was measured using the cells treated in the same manner via injection of glucose (10 mM), oligomycin (1 µM), or 2-DG (100 mM). In addition, the basal cellular glycolytic rate of untreated, curcumin-treated, and WZ35-treated HCCLM3 was calculated.
Flow cytometry analysis for ROS determination
ROS production was quantified by staining the cells for 30 minutes with 10 μM 2,7-dichlorofluo-rescein diacetate (DCFH-DA) in DMEM at 37 °C. The cells were then treated for 2 h with corresponding compounds (10 μg/mL), washed twice with DMEM, and DCFH-DA fluorescence was quantified by BD Accuri TM C6 flow cytometer (BD, Franklin Lakes, NJ).
Detection of glucose in the supernatant
HCCLM3 cells were plated in 3 dishes (60 mm) with DMSO（10 μL/mL）、WZ35 (10 μg/mL) and blank. After a 18 h drug treatment, the supernatant was collected and analyzed. According to Beckman Kurt biochemical analysis system, glucose determination kit (Beckman Coulter, CA, USA) was used to detect the glucose content in the culture medium supernatant. In the presence of adenosine triphosphate (ATP) and magnesium ions, the glucose was phosphorylated by hexokinase (HK) to produce glucose-6-phosphate and adenosine diphosphate (ADP). Glucose-6-phosphate dehydrogenase (G6P-DH) specifically can oxidize glucose-6-phosphate to 6-phosphogluconic acid. Oxidized nicotinamide adenine dinucleotide (NAD+) was also reduced to nicotinamide adenine dinucleotide (NADH). The increase of absorbance at 340 nm was proportional to the glucose concentration in the sample.
Glucose metabolism test
A CCK-8 kit was used to measure the cellular proliferation. HCCLM3 cells were plated in 96-well plates (8x103/well) overnight, medium was changed to sugar-free medium the next day, after which the cells were left untreated or treated with Glucose (1 mM), Glucose (25 mM), 2-DG (1 mM), WZ35 (10 μg/mL). After an 18h-drug treatment, the CCK-8 kit was used to measure the viability, with absorbance at 450 nm being quantified via microplate reader (Spectramax m5, Molecular Devices).
TCA: Targeted metabolite analysis was performed on a Xevo G2-XS QT of mass spectrometry coupled with an ACQUITY UPLC (Ultra Performance Liquid Chromatography) system and data analysis was done with Progenesis QI (all from Waters, Milford, MA, USA). The metabolites in a homogenate of 1×106 cells were extracted using chloroform, methanol, and water at a ratio of 8:4:3 and resuspended in 1％ acetonitrile. For UPLC, the samples were injected on an HSS T3 column (100×2.1 mm, 1.8 μm) using an 8-min gradient containing flow phase A (0.1％formic acid-water) and flow phase B (0.1% formic acid-acetonitrile) at a flow rate of 0.5 ml/min. For the negative ion mode, the capillary energy and sample cone were set as 2000 V and 20 V, respectively. Scan time was set at 0.1 s intervals for 60 s.
Other metabolomics: A. 95% water: 5% acetonitrile + 10 mM ammonium acetate + NH4OH (PH = 10 ); B. 95% acetonitrile : 5% water + 10 mM ammonium acetate + NH4OH ( PH = 10 ). Waters Acquity UPLC BEH Amide 2.1x100 mm, 1.7 μm. Time, flow velocity（mL/min）, A1%, B1% were arranged in the following order: initial, 0.4, 1, 99; 0.1, 0.4, 1, 99; 6, 0.4, 70, 30; 6.5, 0.4, 1, 99; 10, 0.4, 1, 99.
Female BALB/c nude mice (6-8 weeks old; ~20 g) from Wenzhou Medical University Laboratory Animal Center were housed under SPF conditions. These animals were subcutaneously injected with a 100 μL volume containing 5×106 HCCLM3 cells. When tumors grew to 100 mm3, the mice were randomized into 4 groups (n=6/group) that were intraperitoneally administered saline (Mock) vehicle, curcumin (25 mg/kg), or WZ35 (25 mg/kg) daily for 17 consecutive days. Animal body weight and tumor volumes were monitored every other day, with the latter being calculated as follows: tumor volume = length × width × height/2. After 17 days, sodium pentobarbital was used to euthanize mice, and the tumor tissues were collected to make paraffin sections for HE staining. Our project was conducted under guidelines approved by the Experimental Animal Ethics Committee of Wenzhou Medical University（ID number：wydw2019-0446）.
Tissue microarrays (TMAs) and IHC staining
HCC TMAs composed of 110 FFPE tissue sections (4 μm thick) were prepared the same way as previously described by Guttà C et al . TMA IHC staining was conducted based on a standard approach. Briefly, the sections were deparaffinized using xylene, rehydrated with an ethanol gradient, treated to quench peroxidase activity, blocked with 5% normal goat serum, and incubated with anti-YAP/GLUT1 overnight at 4 °C. As a negative control, the antibody was omitted. The slides were then probed with biotin-labeled goat anti-rabbit IgG, treated with a streptavidin peroxidase solution (SABC kit, Boster, Wuhan, China), and then 3,3-diaminobenzidine (Boster, Wuhan, China) in PBS with 0.05% H2O2 was used to treat the samples for 5 min at the room temperature for color development, after which the samples were counterstained using hematoxylin.
IHC staining assessment
Two pathologists blinded to the samples evaluated the clinicopathological information independently and scored IHC images of TMA samples. We utilized the Image-Pro Plus 6.0 (IPP) (Media Cybernetics, MD, USA) to calculate the integrated optical density (IOD) for the stained images, with this value corresponding to total staining per sample area . Entire TMAs were scanned in order to quantify protein levels in the TMA levels. IHC staining was evaluated based on immunoreactive score (IRS) values as described in the prior studies , with these scores reflecting both staining intensity and the percentage of the positive cells. The percent positivity was stained as follows: 1 (≤ 10%), 2 (10-50%), 3 (50-80%), or 4 (≥ 80%). The staining intensity was scored as: 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). These two scores were then multiplied together to yield the IRS value (0 – 12), with these values being deemed either low (≤ 6) or high (>6).
SPSS 19.0 (SPSS Inc., IL, USA) was utilized for statistical analysis. The data has been represented as the means ± SD from three independent experiments, and was compared via Student’s t-tests. P < 0.05 was set as the significance threshold.