High performance liquid chromatography (HPLC)-grade quercetin was purchased (>98%, Sigma-Aldrich, USA) in an anhydrous powdered form. Gold nanoparticles (AuNPs) were synthesized by reducing 1 mM gold chloride with a freshly prepared quercetin solution in absolute alcohol. The pale-yellow solution turned to deep red as the quercetin nanoparticles were formed. PLGA (50 mg) was added to an aqueous dispersion of AuNPs. Next, we added this mixture drop-wise to 20 ml of an aqueous solution with a stabilizer (1% polyoxyethylene-polyoxypropylene; F68). The mixture was stirred at 400 rpm and 4°C until the organic solvent had evaporated completely. The redundant stabilizer was removed by repeated washing and centrifugation (25,000 x g and 4°C for 30 min), and the pellet was then resuspended in Milli-Q water. The quercetin nanoparticles were stored at 4°C for further study. Fluorescent dye was conjugated to the gold surface by adding FITC dye to the PLGA and quercetin nanoparticle mixture, which were performed in the dark. In addition, scanning transmission electron microscopy (SEM) (FEI Quanta650, USA) was used to determine the size of naked quercetin nanoparticles. Further, dynamic light scattering (DLS) with LB-550 DLS particle size analyzer (Horiba Scientific, Edison, NJ, USA) was used to examine the average size of quercetin nanoparticles. We analyzed the data in the automatic mode. Size is presented as the mean value of 20 runs, with triplicate measurements for each run. We measured the zeta potential of the quercetin nanoparticles in the same instrument using the same procedure.

The human liver cancer cell lines of MHCC97H, Hep3B, HCCLM3 and Bel7402 were obtained from American Type Culture Collection (ATCC, VA, USA). Cells were cultured as monolayers in RPMI-1640 culture media supplemented with 10% heat-inactivated fetal bovine serum, 1×10⁵ U/l streptomycin sulfate, pH 7.2 (Gibco Corp., Gaithersburg, MD, USA) with a concentration of 1×10⁶/ml at 37°C, 5% CO₂ at 37°C.

Liver cancer cell viability was assessed using MTT assay (Roche Diagnosis, IN, USA). In brief, liver cancer cell lines were planted at 4×10³ cells/well in 96-well plates. Cells were then cultured overnight, and next the cells were changed into fresh medium with various doses of quercetin nanoparticles dissolved in DMSO with final concentration of 0.1%. After incubation for 48 h, the cell growth was measured. The cell viability was assessed as the percent cell viability compared to the vehicle-treated control cells without quercetin nanoparticles administration, which were determined arbitrarily as 100% viability.

One hundred liver cancer cells per well in 60-mm plates were cultured in 10% FBS DMEM. Cells were treated with quercetin nanoparticles of the indicated concentrations for 24 h. After another 7 days of incubation, the cell colonies were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min and then stained by Giemsa for 30 min. Each clone with >50 cells were evaluated. Clone forming efficiency for cells was calculated based on: Plate colony formation inhibitory ratio = (number of colonies treated with quercetin nanoparticles / number of cells inoculated) × 100%.

Wound-healing assays were carried out using migration culture dish inserts. Liver cancer cells were seeded in the chambers of the culture dish insert and transfected. Forty-eight hours after transfection, the insert was removed and fresh culture medium was added to start the migration process. Cells were treated with indicated doses of quercetin nanoparticles in full medium and kept in a CO₂ incubator. After 48 h, medium was replaced with PBS, and images were acquired using a Zeiss Axiovert 24 light microscope and an Axiocam MRc camera.

Flow cytometric assay was used to clarify cell apoptosis. The cells were collected with trypsinisation and then washed twice with PBS, and fixed in cold 80% ethanol, and finally stored at 4°C overnight. The cells were washed with PBS twice and RNase A (10 mg/ml) was administered for analysis. Propidium iodide was then added to tubes at a concentration of 0.05 mg/ml and then incubated for 20 min at 4°C in the dark. FITC-labeled Annexin V/PI staining was applied according to the manufacturer's instructions (Keygen, Nanjing, China). In brief, 1×10⁶ cells in each well were suspended with buffer containing FITC-conjugated Annexin V/PI. Samples were then analyzed via flow cytometry.

For western blot analysis, sample tissues and cells were homogenized into 10% (wt/vol) hypotonic buffer (25 mM Tris-HCl, pH 8.0, 1 mM EDTA, 5 µg/ml leupeptin, 1 mM Pefabloc SC, 50 µg/ml aprotinin, 5 µg/ml soybean trypsin inhibitor, 4 mM benzamidine) to yield a homogenate. Additionally, the final supernatants were obtained by centrifugation at 12,000 rpm for 20 min. Protein concentration was determined by BCA protein assay kit (Thermo, USA) with bovine serum albumin as a standard. The total protein extract will be used for western blot analysis. Equal amounts of total protein of tissues were subjected to 10 or 12% SDS-PAGE followed by immunoblotting using the primary polyclonal antibodies (Table I). Immunoreactive bands were visualized by ECL Immunoblot Detection system (Pierce Biotechnology, Inc., Rockford, IL, USA) and exposed to Kodak (Eastman Kodak Co., USA) X-ray film. Each protein expression level was defined as grey value (Version 1.4.2b, Mac OS X, ImageJ, National Institutes of Health, USA) and standardized to housekeeping genes (GAPDH) and expressed as a fold of control.

Total RNA from the cultured cancer cells was obtained by the miRNA Isolation kit (Sigma, USA) according to the manufacturer's instructions. Then the cDNA was synthesized from RNA. Real-time PCR was conducted with the Applied Biosystems 7500 Sequence Detection system by the use of iQ™ SYBR Green Supermix (Bio-Rad Laboratories, USA) with 5 ng cDNA and 10 pM related primer. The cycling condition was conducted at 94°C for 60 sec; followed by 45 cycles at 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec; followed by 95°C for 10 sec, 65°C for 45 sec, and 40°C for 60 sec. The data were normalized to the housekeeping gene GAPDH and U6 small nuclear RNA expression and calculated as 2^−ΔΔCT expression. The primers used are shown in Table II.

The cells grown on chamber slides were washed in PBS and fixed for 15 min at room temperature with 4% paraformaldehyde, followed by 20 and 30% sucrose dehydration for 24 h each. Then, the samples were incubated with primary antibodies (Cyto-c, P50 and NF-κB, Cell Signaling Technology, USA) at 4°C overnight after deparaffinized and rehydrated. Fluorophore-conjugated secondary antibodies were treated for 1 h at 25°C thermostat. The Alexa Fluor 488 labeled anti-rabbit secondary antibodies (Invitrogen, CA, USA) and DAPI (Sigma-Aldrich) were used in this part. Samples were then subjected to immunofluorescence staining via epifluorescence microscopy (Sunny Co.). Leica TCS SP5 confocal microscope (Leica, Richmond Hill, ON, Canada) was used to obtain images and carried on blinded with respect to treatment groups. ImageJ 'measure' tool analyzed fluorescence intensity through examining mean intensity of each selected areas (a minimum of 10 rectangles).

The transfection of targeted siRNAs or expression vectors were conducted by Lipofectamine 2000 reagent based on the manufacturer's protocol (Invitrogen).

Streptavidin-agarose pull-down assay was used to determine the binding of AP-2β, p50 to hTERT or COX-2 core promoter probes. A biotin-labeled double-stranded probe corresponding to hTERT and COX-2 promoter sequence was synthesized. The binding assay was applied by mixing 4 µg biotinylated DNA probe, 400 µg nuclear extract proteins and 40 µl 4% streptavidin-conjugated agarose beads at room temperature for an hour in a rotating shaker. Beads were then pelleted via centrifugation in order to pull down the DNA-protein complex. After washing, proteins in complex were evaluated through immunoblotting with antibodies (1 µg/ml of each sample) specific for AP-2β and p50. The mixture was then incubated at room temperature for 1 h with shaking, and then centrifuged to pull down the DNA-protein complex. DNA-bound AP-2β and p50 protein was dissociated and analyzed by western blotting. Non-immune rabbit IgG (1 µg/ml) was used as negative controls.

The mouse experiments were conducted in the Animal Laboratory Center. MHCC97H cells (1×10⁷ cells) treated with or without quercetin nanoparticles were suspended in 100-µl serum-free medium and injected subcutaneously into the left flank of 4- to 6-week old male BALB/c nu/nu nude mice. After two weeks, when the tumor diameters reached 3×4 mm, the tumor cell-inoculated mice were divided into four treatment groups randomly, the control (Con) treated with PBS; the quercetin nanoparticle groups treated with 30, 40 and 50 mg/kg, respectively, by intraperitoneal injection every day. Tumor size was measured with digital caliper and calculated as V = LS2/2 (L is the longest diameter and S is the shortest diameter). Tumor volume and animal weight were measured twice every seven days, and at ~5 weeks after treatment, mice were sacrificed. Body weights were also recorded. Tumors were excised, weighted, fixed in 10% neutral formalin, and embedded in paraffin for histological analysis.

The xenograft tumors were performed for hematoxylin and eosin staining. In brief, fresh tissues were fixed in paraffin, and for immunohistochemistry, the fresh tumor tissues were fixed in formalin for 48 h. Then the tissue block was put in paraffin and next cut into the desired thickness with a microtome, and was then fixed into a slide. After washing, the sections were prepared for blocking and incubating with antibodies, including AP-2β, TUNEL and COX2, which were diluted 1:100 in 5% horse serum with PBS at 4°C overnight. Sections were then incubated with diluted streptavidin-peroxidase HRP conjugates at room temperature by a staining kit, according to the manufacturer's instructions. The sections were then stained with hematoxylin for 3 min and mounted and analyzed under a phase-contrast microscope.

Data were expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad PRISM (version 6.0; Graph Pad Software) by ANOVA with Dunnet's least significant difference post hoc tests. A p-value <0.05 was considered statistically significant.

The quercetin nanoparticle surface morphology was explored by scanning electron microscopy (SEM) (Fig. 1A). The images displayed spherically-shaped quercetin nanoparticles with a smooth surface and without pinholes or cracks. The dynamic light scattering (DLS) data indicated that the mean quercetin nanoparticle diameter was 106.7 nm (Fig. 1B). Additionally, the zeta potential was −19.1 mV (Fig. 1C). In this experiment, four liver cancer cell lines, including MHCC97H, Hep3B, HCCLM3 and Bel7402, were chosen to explore whether quercetin nanoparticle was effective on liver cancer inhibition. Hence, a 24-h dose-dependent (0–60 µg/ml) study with MHCC97H, Hep3B, HCCLM3 and Bel7402 cells was conducted. As shown in Fig. 1D, the liver cancer cell viability decreased. Additionally, quercetin nanoparticle was much more toxic to MHCC97H cells than the other cells. Therefore, the following experiments were performed with MHCC97H cells.

We first evaluated the effects of quercetin nanoparticle on MHCC97H cell growth. As shown in Fig. 2A, treatment with quercetin nanoparticle inhibited colony formation in a dose-dependent manner. We next analyzed the effect of quercetin nanoparticle on changes in cell morphology and spreading in MHCC97H cells. As shown in Fig. 2B, the cells treated without quercetin nanoparticle contributed to a cell layer. In addition, more spread and filopodia was observed. In the contrast, treatment with quercetin nanoparticle significantly promoted the cell-to-cell contact and reduced the cell spreading with lower filopodia formation compared with control group, suggesting that quercetin nanoparticle enhanced alterations in MHCC97H cell morphology and spreading. Further, wound-healing assay was used to determine the role of quercetin nanoparticle in MHCC97H cell migration. Consistently, after making a scratch the gap and wounding space between MHCC97H cell layers was occupied partially due to the migrating cells after 48 h in the control group. However, the empty space was not occupied by the MHCC97H cells after quercetin nanoparticle treatment, suggesting that the liver cancer cell migration was inhibited markedly after quercetin nanoparticle administration, which was dose-dependent (Fig. 2C). To further identify the possible mechanisms related to cell migration, we assessed the P-27, c-Myc, cyclin-D1, CDK1, MMP7 and β-catenin protein levels. The results showed that P-27 was expressed highly after quer-cetin nanoparticle treatment, displaying antitumor activity via P-27 upregulation. However, c-Myc, cyclin-D1, CDK1, MMP7 and β-catenin, important factors promoting cell cycle, were significantly downregulated after quercetin nanoparticle treatment (Fig. 2D).

The effect of quercetin nanoparticles on apoptosis in MHCC97H cells was studied. Treatment with quercetin nanoparticle at different doses of 30, 40, and 50 µg/ml was performed on the liver cancer cells (Fig. 3A). Compared to the control group, treatment with quercetin nanoparticle significantly upregulated the number of apoptotic cells (Fig. 3A). Caspase cascade activation forms the essential basis for apoptosis. Cytochrome c releasing from the mitochondrial inter-membrane space into the cytoplasm is the precondition of caspase-dependent apoptosis pathway. Thus, we measured the expression of pro-apoptotic proteins, including caspase-9, caspase-3, and Cyto-c in MHCC97H cells via western blot analysis. Treatment with quercetin nanoparticle promoted the upregulation of the cleaved caspase-9, caspase-3 and cytoplasm Cyto-c effectively compared with the control group (Fig. 3B), indicating the effect of quercetin nanoparticle on apoptosis induction in liver cancer cells. Immunofluorescence imaging (IFI) was also performed to observe the changes of subcellular localization of Cyto-c in MHCC97H cells to explore whether quercetin nanoparticle could stimulate Cyto-c release. Treatment with quercetin nanoparticle at different concentrations induced Cyto-c release from the inter-mitochondrial space into the cytoplasm (Fig. 3C). The results above demonstrated that quercetin nanoparticle might enhance the caspase activation via Cyto-c-dependent apoptosis in liver cancer cells.

hTERT is a mark of tumorigenesis, which is highly modulated by transcriptional factor AP-2β (19). In order to determine if quercetin nanoparticle influenced the AP-2/hTERT signaling pathway in liver cancer cells, MHCC97H cells were administrated with quercetin nanoparticles, and the protein and mRNA expression of AP-2β and hTERT were examined via western blot and RT-PCR assays. Treatment with quercetin nanoparticles reduced the hTERT and AP-2β protein and mRNA levels compared to the control group (Fig. 4A and B). hTERT expression is closely related to the AP-2β binding activity on hTERT promoter. Next, streptavidin-agarose pull-down assay was performed to test the effect of quercetin nanoparticle on AP-2β binding activity in MHCC97H cells. Treatment with quercetin nanoparticle had a potential role in suppressing AP-2β protein levels (Fig. 4A), thus suppressing the binding of AP-2β to hTERT promoter (Fig. 4C). Further, we also explored the role of quercetin nanoparticle in regulating hTERT promoter activity. The results suggested that treatment with quercetin nanoparticle effectively inhibited hTERT promoter activity (Fig. 4D). Finally, in order to further clarify that the AP-2β signaling is associated with the promotion of cell growth inhibition, MHCC97H cells were transfected with 100 nM AP-2β siRNA or AP-2β-expressing vector and next cotreated with quercetin nanoparticle (50 µg/ml). As shown in Fig. 4E, compared with the non-specific AP-2β siRNA control (si-NC), AP-2β knockdown (si-AP-2β) slightly reduced the cell growth regulated by quercetin nanoparticles. Of note, AP-2β overexpression through AP-2β transfection significantly upregulated the liver cancer cell growth compared to transfection with control vector (CV) (Fig. 4E). The data above illustrated that the promotion of growth suppression by quercetin nanoparticle was regulated, at least partly, through AP-2β/hTERT signaling pathway inhibition in MHCC97H cells.

COX-2 signaling is related to cancer cell growth, invasion, migration and proliferation (20,21). In this regard, we determined the role of quercetin nanoparticle in regulating COX-2 protein levels in MHCC97H cells via western blotting. Treatment with quercetin nanoparticle significantly suppressed COX-2 protein levels (Fig. 5A and B). To prove that quercetin nanoparticles promoted COX-2 signaling inhibition, MHCC97H cells were administered with COX-2-selective inhibitor (COX2-Inh) (20 µM), and then treated with quercetin nanoparticle (50 µg/ml). Treatment with COX-2-selective inhibitor inhibited MHCC97H cell viability (Fig. 5D). However, a combination with quercetin nanoparticle did not affect cell viability suppression significantly regulated by COX-2 inhibitor, suggesting that COX-2 signaling pathway was linked with quercetin nanoparticle-regulated liver cancer inhibition. In addition, COX-2 expression was related to the p50 binding activity on the COX-2 promoter structure. Subsequently, we attempted to explore whether quercetin nanoparticle suppressed the binding of p50 to COX-2 promoter in MHCC97H cells. Streptavidin-agarose pull-down assay results indicated that treatment with quercetin nanoparticle significantly suppressed p50 binding on COX-2 promoter in comparison with the control group (Fig. 5C). The p50 protein levels were not significantly affected by quercetin nanoparticle (Fig. 5A and B).

NF-κB translocation in cell nuclei and cytoplasm plays an important role in modulating COX-2 expression (22). NF-κB activation was regulated highly by its upstream signals, including IKKα and IκBα (23). Fig. 5A shows that quercetin nanoparticle treatment could reduce the phosphorylated IKKα and IκBα, leading to the alteration of NF-κB activation. Immunofluorescence assay was performed to explore the role of quercetin nanoparticle in p50 and NF-κB translocation through a confocal microscope. Treatment with different concentrations of quercetin nanoparticles caused translocation of NF-κB and p50 from the cell nuclei into cytoplasm (Fig. 5E). The results illustrated that the enhanced suppression of liver cancer cell growth by quercetin nanoparticle might be also regulated, at least partly, through the p50/NF-κB/COX-2 pathway in liver cancer cells.

PI3K/Akt and Raf/ERK1/2 signaling pathways play essential roles in cancer progression and are involved in the cancer-related gene regulation, including hTERT and COX-2 (24). To determine whether PI3K/Akt and Raf/ERK1/2 signaling pathways were related to quercetin nanoparticle-regulated liver cancer cell growth suppression, we then explored the effect of quercetin nanoparticle on Akt and ERK activity in MHCC97H cells through western blotting. Quercetin nanoparticle treatment downregulated the phosphorylated Akt and ERK1/2 (Fig. 6A and B). Additionally, the total Akt and ERK1/2 protein levels were not significantly affected by quercetin nanoparticle.

In order to further confirm that quercetin nanoparticle could regulate Akt and ERK pathways to suppress MHCC97H cell growth, the effect of Akt or ERK1/2-selective inhibitor on quercetin nanoparticle-modulated suppression of cell viability in MHCC97H cells was calculated. Treatment with Akt inhibitor (Akt-Inh) and ERK1/2 inhibitor (ERK1/2-Inh) downregulated cell viability effectively (Fig. 6C and D). The data above revealed that Akt/ERK1/2 signaling pathways were important targets for quercetin nanoparticles in suppressing MHCC97H cell growth.

To confirm the role of quercetin nanoparticle in liver cancer growth inhibition, we explored the effects of quercetin nanoparticle on tumorigenicity using an MHCC97H xenograft mouse model in vivo. After administration with quercetin nanoparticles for 35 days, the tumor volumes (Fig. 7A and B) and tumor weights (Fig. 7C) were suppressed significantly by treatment with quercetin nanoparticles. The quercetin nanoparticle treatment did not influence the body weight significantly of the mice (Fig. 7D). These results above supported that quercetin nanoparticle could suppress the xenografted human liver cancer cell growth and proliferation without remarkable adverse effects.

In addition, the IHC staining further illustrated that the tumors in the quercetin nanoparticle-treated groups expressed much lower AP-2β and COX2 levels in comparison to the control group (Fig. 8A and B). In contrast, TUNEL levels were upregulated significantly after quercetin nanoparticle administration, indicating apoptosis was induced by quercetin nanoparticle (Fig. 8A and B). The molecular mechnism by which quercetin nanoparticles inhibited liver cancer progression was explored. As shown in Fig. 8C and D, the Cyto-c/caspase, P50/NF-κB, AP-2β/Htert and Akt/ERK1/2 signaling pathways were inhibited by quercetin nanoparticle administration. As shown in Fig. 8C, the cleaved caspase-9, cleaved caspase-3 and cytoplasm Cyto-c were downregulated significantly after quercetin nanoparticle treatment. Further, the phosphorylated IKKα, IκBα and NF-κB were reduced in the quercetin nanoparticle-treated groups. In addition, P50 was not altered significantly by quercetin nanoparticle treatment, which was consistent with previous results in vitro (Fig. 8D). Also, AP-2β and Htert were decreased after quercetin nanoparticle administration (Fig. 8E). Finally, the reduced p-Akt, Raf and p-ERK1/2 further clarified that quercetin nanoparticle had an inhibitory role in liver cancer progression in vivo (Fig. 8F). Our data above indicated that quercetin nanoparticle could suppress liver cancer development via inhibition of Cyto-c/caspase, P50/NF-κB, AP-2β/Htert and Akt/ERK1/2 signaling pathways.