Quercetin shows anti‐tumor effect in hepatocellular carcinoma LM3 cells by abrogating JAK2/STAT3 signaling pathway

2.1. Reagents

Quercetin, AG490, IL‐6 and DMSO were obtained from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS), trypsin, and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA). Anti‐N‐cadherin, anti‐Vimentin, anti‐E‐cadherin, anti‐MMP‐9, and anti‐Cyclin B1 antibodies were purchased from Abcam (Cambridge, UK). Anti‐Bax, anti‐LC3, anti‐Beclin1, anti‐PCNA and anti‐STAT3, anti‐phospho‐STAT3, anti‐total‐JAK2, anti‐phospho‐JAK2 antibodies were obtained from Cell Signaling Technology (Danvers, MA). Antibody to anti‐P62 was obtained from Proteintech (Chicago, IL).

2.2. Cell lines and cultures, and morphological analyses

The HCC cell line LM3 was purchased from the Chinese Academy of Sciences Committee Type Culture Collection Cell Bank (Beijing, China). These cell lines were cultured in high glucose DMEM (DMEM‐h) supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified incubator at 37°C in 5% CO₂.

For morphology experiments, cells were cultured with indicated treatments for 48 hours. Cell morphology was observed using a phase‐contrast microscope and imaged (400×).

2.3. Cell viability

HCC cell line LM3 was plated at a density of 4 × 10⁴ cells/ml in 96‐well plates (100 µL medium per well) with five replicates. HCC cells were treated with QE at doses of 0, 20, 40, 60, 80, 100,120, 140, 160, and 200 µmol/L. After seeding for 24, 48, and 72 hours, cell proliferation and viability were measured using the CCK8 assay (Dojindo Laboratories, Tokyo, Japan) according to the manufacturer's instructions.

2.4. Flow cytometry analysis

HCC cells in logarithmic growth phase were seeded into 6‐well plates and exposed to QE (40, 80, and 120 µmol/L) for 48 hours. The cells were digested and washed with phosphate‐buffered saline (PBS) for two times. The pellets were suspended in 1× binding buffer, and incubated with annexin‐V/propidium iodide (PI: BD Biosciences, San Jose, CA) following the standard steps. The samples were examined using flow cytometry (Cytomics FC500; Beckman Coulter, Fullerton, CA).

After similar processing, the cells were fixed with 75% ethanol at −20°C overnight, and then resuspended in PBS containing 20 mg/mL PI and 50 mg/mL RNase A. The cells were then incubated in the dark for 30 minutes at 4°C and the cell cycle was analyzed using flow cytometry.

2.5. Colony formation assay

After resuspension, LM3 cells were plated into 6‐well plates and were treated with vehicle or QE (80 and 120 µmol/L). After 14 days, the colonies developed in each well were photographed and counted.

2.6. Wound‐healing assay in vitro

The LM3 cells were planted in 6‐well plates, and when the cell fusion reached 80%, we used the tip of a 200 mL pipette to cut the cells' surface and form a wound. After the cells were washed three times, they were treated with DMEM (without FBS) or QE (80 and 120 µmol/L) and incubated for 36 hours to quantify the wound healing process. The distance of the scratch closure was examined at 12, 24, and 36 hours. At least ten microscopic fields were analyzed, and representative figures are shown.

2.7. Transwell invasion assays

Experiments were performed using a Transwell chamber (BD Biosciences). Basement Membrane Matrix (Corning, Corning, NY) was placed into the upper chamber. DMEM‐h containing 10% FBS was placed in the lower chamber. 1 × 10⁵ cells (LM3) in serum‐free medium were placed in the upper chamber with different concentrations of QE. After 48 hours, the cells attached to lower membrane surface were stained with 0.05% Crystal Violet.

2.8. Confocal microscopy

The cells were seeded onto glass cover slips. After 48 hours, treated and untreated cells were washed with PBS and then immersed in the fixation solution. These fixed cells were incubated with antibodies overnight at 4°C. Immunofluorescence was detected with anti‐rabbit immunoglobulin G (IgG) conjugated with Alexa Fluor 488 (green) and DAPI (blue) (Invitrogen, Carlsbad, CA) was used for nuclear staining. The immunofluorescence was observed using a confocal microscope (Olympus, Tokyo, Japan).

TUNEL staining was performed using an EdUTP TUNEL cell detection kit (Ribobio, Gangzhou, China) according to the manufacturer's instructions, and was observed using a confocal microscope.

2.9. Animal experiments

Animal experiments were performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Shanghai Tongji University, China.

Ten nude mice were raised in an environment meeting international standards and were monitored daily. LM3 HCC cells were subcutaneously injected into the upper lateral area of mice at the density of 5×10⁶/100μL. After 1 week, the subcutaneous tumor tissue grew to the size of about 1.0 mm³. All mice were randomly divided into two groups (five mice in each group) according to whether they received QE intervention or not.

QE was suspended in saline and administered for 21 days by gavage at the dose of 100 mg/kg (200 µL for every model mouse). Tumor volume and mouse weight were measured every 3 days and the mice were anesthetized and euthanized. Tumor tissues were harvested for further analysis.

2.10. Hematoxylin and eosin staining

A portion of tumor tissue was fixed with 4% paraformaldehyde and embedded into wax blocks. Sections (5 μm thick) were cut, and then were dewaxed and hydrated. Were stored at room temperature. Hematoxylin and eosin (H&E) was added to stain the nuclear regions and cytoplasm. Histopathological changes were observed under a light microscope.

2.11. TUNEL (terminal deoxynucleotide transferase dUTP nick end labeling) assays

Apoptosis of tumor tissues was assessed using the TUNEL assay. Paraffin‐embedded sections (5 µm) were cut and mounted on glass slides. After treatment according to the instructions, sections were incubated with the TUNEL reaction mixture for 1 hour at 37°C. Sections were observed under a light microscope.

2.12. Protein extraction and western blotting

Total protein from cells or tissues was isolated using radioimmunoprecipitation assay buffer. The protein obtained from supernatants was quantified according to the standard protocol. Proteins were separated using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride or nitrocellulose membranes. After incubation with primary antibodies and secondary antibody, blots were developed by using the Odyssey Two‐color Infrared Laser Imaging System (Li‐Cor, Lincoln, NE). The signal generated by β‐actin was used as an internal control.

2.13. RNA extraction and quantitative real‐time (qRT)‐PCR analysis

Total RNA was extracted by using TRIzol reagent according to the standard protocol and first‐strand cDNA was synthesized using the reverse transcription kit (TaKaRa Biotechnology, Dalian, China). The cDNA was used in real‐time PCR reactions to analyze indicators expression. Primers used in the PCR reactions are listed in Table 1. The real‐time PCR experiments were performed according to the protocol of the real‐time PCR kit (Takara, Otsu, Shiga, Japan). The expression of those genes was calculated using the 2^{(‐ΔΔC(T))} method.

2.14. Statistical analysis

All the experiments were conducted three times and were analyzed using Graph Pad Prism 5.0 software. Comparisons among multiple groups were conducted using one‐way ANOVA with the Student's t test to compare between two groups. A value of P < 0.05 was considered statistically significant.

3.1. QE suppressed LM3 cell viability and induced LM3 cell apoptosis

The HCC cell line, LM3, was intervened by QE (0, 20, 40, 60, 80, 100, 120,160, and 200 μmol/L) for 24, 48, and 72 hours, whose survivability was measured using CCK8 kits. Cell growth curves were constructed on the basis of data obtained. The analyzed results showed that QE played an inhibition role of the viability of LM3 cells varing with dose and time. We calculated the half maximal inhibitory concentration at 48 hours, which is shown in Figure 1A. QE also exhibited typical morphological changes in LM3 cells. We selected effective QE concentrations (80 and 120 μmol/L) for treatment of LM3 cells for the following experiments.

To assess the extent of apoptosis induced in LM3 cells after QE treatment, flow cytometry, western blotting, qRT‐PCR, colony formation assays, and immunofluorescence were performed. Apoptotic cell death was divided into early stage apoptotic cell death and late stage apoptotic cell death, which are marked as annexin‐V+/PI− and annexin‐V+/PI+. And cell death caused by apoptosis was quantified by the percentage of them. The results demonstrated a growth in the proportion of early stage apoptotic cells in a concentration‐dependent manner after treatment with QE (Figure 1B). We collected protein and RNA from cells treated with QE (0, 80 and 120 μmol/L for 48 hours), and determined the protein and gene expression levels. PCNA is an index associated with DNA synthesis, and can reflect the proliferation of cells. Bax is a classical index for promoting apoptosis. Figure 1D,E exhibit the blots and data; they show that QE reduced the expression of PCNA, and increased the expression of Bax. The results in Figure 1F show that QE suppressed the formation of colonies. In addition, the cleaved DNA in apoptotic cells combined with the TUNEL reagent and showed bright green fluorescence. Figure 1G shows that QE increased the fluorescence intensity of TUNEL. After these cancer cells were treated with QE at doses of 0, 80, and 120 μmol/L for 48 hours, we performed PI staining to measure the distribution of cell cycle. The results reveal that QE treatment induced cells were arrested in the S and G2/M phases, and the number of G0/G1 phase cells was reduced (Figure 1C). Moreover, the protein expression level of cyclin B1, a cell cycle‐related protein, was decreased by treatment with QE as shown by western blotting (Figure 1D).So, we concluded that the inhibition effect of QE in cell proliferation may have a relationship with the cell cycle arrest.

3.2. QE inhibited LM3 cell migration and invasion

We then treated LM3 cells with QE at concentrations of 0, 80, and 120 µmol/L for 48 hours, and detected the mRNA levels of important epithelial‐mesenchymal transition (EMT) biomarkers, E‐cadherin, vimentin, and matrix metallopeptidase 9 (MMP9). The results showed that QE escalated mRNA expression levels of E‐cadherin, and reduced mRNA expression of vimentin and MMP9 with QE concentrations increasing (Figure 2A). Furthermore, we performed western blots to detect the protein levels of N‐cadherin, E‐cadherin, vimentin and MMP9 (Figure 2B). Immunofluorescence assays were performed to set out the protein levels of E‐cadherin, vimentin and MMP9, and we found that the results were in line with the trend obtained in previous experiments (Figure 2C). From what has been discussed above, our results revealed that QE reversed the EMT process of LM3 cells. Then, we performed transwell invasion (Figure 2D) and wound healing (Figure 2E) assays to testify the inhibition effect of QE on HCC cells' migration and invasion. The observed results expressed that invasion and migration were significantly restrained by treatment with QE.

3.3. QE promoted HCC autophagy

Autophagy is the process of cell phagocytosis of cytoplasmic proteins or organelles and degradation, to meet the requirement for renewal of some organelles and the metabolism of cells. We explored whether QE could activate autophagy in HCC cells. Beclin‐1, LC3 and P62 are typical biomarkers for autophagy. As shown in Figure 3A, significantly up‐regulated expression of LC3, and down‐regulated expression of P62 were observed after QE treatment in a dose and time‐dependent manner. Furthermore, we treated cells with QE (80 μmol/L) and analyzed the proteins at 0, 6,12, and 24 hours after QE treatment using western blot analyses. The results were in accordance with the qRT‐PCR results, which are shown in Figure 3B. The results of immunofluorescence assays showed the up‐regulation of LC3 by QE treatment (Figure 3C). Taken together, QE induced autophagy in HCC cells.

3.4. QE inhibited the activation of JAK2/STAT3 pathway

QE is able to affect various signaling pathways, for example, the STAT3 pathway. To make sure whether QE could modulate the activation of the STAT3 pathway, we detected the expression of P‐STAT3 in LM3 cells by immunofluorescence staining. The results shown in Figure 4A suggested that QE reduced the expression of p‐STAT3, and therefore, we hypothesized that the antitumor effect of QE was related with the STAT3 pathway. AG490 and IL‐6 were confirmed that they could take an inhibitor or promoter role in JAK2/STAT3 signaling in various cells. And then, we used AG490(50μM), IL‐6(100ng/ml) and QE(80μM) to treat LM3 cells ,and collected their protein. The changed expression of STAT3 and JAK2 was as shown in Figure 4B. We found that there was no obvious change of the STAT3 and JAK2 expression in cells after treatment with AG490, IL‐6 or QE. However, the expression changes of phosphorylated STAT3 and JAK2 induced by AG490 could be enhanced by QE, and QE could reverse the changed expression caused by IL‐6. In conclusion, QE could inhibit the activation of the JAK2/STAT3 pathway effectively.

Then, we used qRT‐PCR, WB, wound healing and immunofluorescence staining to confirm whether QE could reverse STAT3 signaling induced apoptosis, metastasis and autophagy. The results of above experiments were exhibited in Figure 4C‐F, which testified our hypothesis. QE significantly showed opposite effect of IL‐6, and could weaken its influences. The changes caused by IL‐6 on PCNA, P62 and MMP‐9 protein expression were changed by QE, and images of immunofluorescence staining show the same trend. Besides, the speed of wound healing was expedited by IL‐6 and retarded by QE. Briefly speaking, QE could reverse the changes induced by STAT3 pathway, and that is to say, the antitumor effect of QE at least partly depended on the STAT3 signaling pathway.

3.5. QE inhibited LM3 cell tumor growth in an animal model

Figure 5A shows that QE has no obvious adverse effect on the liver. Several nude mice were given an injection of LM3 cells subcutaneously, and then received saline or QE (100 mg/kg) treatment to evaluate the in vivo effect of QE for HCC progression. QE treatment significantly downsized tumor volume compared to the control group by approximately 70% (Figure 5B), and mouse weights and tumor volume were reduced with time (Figure 5C). H&E staining of tumor tissues showed obvious necrosis in the QE‐treated group. Section staining of tumor tissues analyses showed more TUNEL‐positive cells in the QE group, while being fewer in the vehicle group (Figure 5D). Besides, to clarify the proapoptosis effect of QE in cancer tissues, we measured the expression of PCNA and Bax using western blot. The expression of PCNA was effectively reduced by QE treatment, and Bax levels were increased. Also, the expression of Beclin‐1 was changed in line with in vitro experiments. Together, these results suggested that QE inhibited tumor growth in vivo. (Figure S1)