Methyl gallate (purity, ≥98%; molecular weight, 184.15; formula, C₈H₈O₅) was obtained from Sigma-Aldrich (St. Louis, MO, United States). Methyl gallate stock solution was prepared using dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, United States) and stored at −20°C. The final concentration of DMSO in the cell culture medium was less than 0.1% in all treatments.

Human hepatocellular carcinoma cell lines BEL-7402 and human normal hepatocytes LO2 were received from Fu Heng BioLogy (Shanghai, China). BEL-7402 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) (Sangon Biotech Co.,Ltd., Shanghai, China), and 1% penicillin/streptomycin (P/S) at 37°C in a humidified atmosphere of 5% CO₂. LO2 cells were cultured in DMEM medium, and the conditions were consistent with BEL-7402 cells.

Cell viability was measured using MTT assay. BEL-7402 cells were seeded in 96-well plates at 8,000 cells per well and treated with different concentrations of MG (20, 40, 80, 160, 320, and 640 μM) and 5-Fluorouracil (5-FU, 50 μM). After the specified time, 10 μL MTT (5 mg/ml) was added, incubated for 4 h at 37°C. The supernatant was discarded, and 150 µL of DMSO was added to every well and rotated for 10 min to dissolve the purple formazan crystals formed. Absorbance was measured on an microplate reader (AMR-100, Allsheng, China) at 490 nm.

Inoculate 1,000 cells per well into 6-well plates overnight. Different concentrations of MG (0, 40, 80, 160 μM) and 5-FU (50 μM) were added to the medium. The medium is replaced every 5 days. After 2 weeks of culture, The cells were fixed using 4% paraformaldehyde (PFA) and then stained with 0.5% crystal violet (Meilunbio, Dalian, China). Wash 6-hole plate several times with ddH₂O, dry and photograph.

BEL-7402 cells were seeded into 6-well plates at 5×10⁵ cells per well, were incubated until 80–90% confluency. Using a sterile 200 μL pipette tip, a scratch wound was created by scraping the cell monolayer. BEL-7402 cells were washed three times with PBS, and the broken cells were washed. Then the cells were cultured in serum-free medium containing different concentrations of MG, and the images were obtained under microscope at 0, 24 and 48 h. The wound healing area was analyzed by Image-Pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD).

The invasion was measured by Matrigel invasion chamber (Corning, NY, United States) with 8 μm pore size membrane. The bottom of the superior ventricle was precoated with 1 mg/ml 60 μL Matrigel (Lu et al., 2016). BEL-7402 cells were seeded (4 × 10⁵ cells/ml 200 µL) into the upper transwell chambers with serum-free RPMI1640. The RPMI1640 medium containing 20% FBS was added as a chemical inducer in the lower room. After incubation for 24 and 48 h, the cells in the upper room were gently scraped with cotton swabs, and the cells invading the submembrane surface were fixed and stained. Take pictures in 6 randomly selected fields of view under microscope (Nikon Eclipse E100, Japan).

RIPA buffer (Beyotime, China) was used to lyse cells to obtain total protein, and the protein concentration was determined by BCA method. The same amount of samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein was transferred to the PVDF membrane (Millipore, Burlington, United States). After blocking with 5% skim milk powder (Beyotime, China) for 2 h at room temperature, the membrane and antibody were incubated overnight at 4°C. The primary antibodies were as follows: AMPK, NF-κB, p-NF-κB, MMP9, MMP2, TIMP-2, E-cadherin, Vimentin and β-actin (Beyotime, China). Then incubated with HRP-labeled Goat Anti-Rabbit IgG (H + L) (Beyotime, China) at room temperature for 2 h. Protein bands were detected by enhanced chemiluminescence (Tanon chemiluminescent gel imaging system, Tanon-5200Multi, China).

All animal experiments were approved by the Ethics Committee of the Guangdong Pharmaceutical University. Four-week-old male BAL B/C nude mice were procured from Guangdong Medical Laboratory Animal Center. After 1 week of acclimatization, 0.2 ml of BEL-7402 cells (1 × 10⁷ cells/ml) were subcutaneously injected into the right axillary region of the mice, followed by drug administration when tumor diameter was >3 mm. For drug treatments, mice were randomly divided into six groups. The control group was administered saline intraperitoneally (ip), the positive group was administered 5-FU (15 mg/kg, ip, three times a week), three experimental groups were administered different concentrations of MG (40, 80, 160 mg/kg/d, intragastric; ig), and the combination group was administered 5-FU and MG (5-FU, 15 mg/kg, ip, three times a week; MG, 80 mg/kg/d, ig). Tumor volume and body weight were measured every 3 days. Tumor volume was calculated as follows: (width² × length)/2. After 21 days of drug administration, the mice were euthanized, and the xenografts, liver, and spleen were dissected and weighed. The average tumor weight, tumor inhibition rate (%), and organ weight ratio were calculated. Tumor inhibition rate (%) calculated by tumor weight. The tumor inhibition rate was calculated as: (control group tumor average weight—experimental group tumor average weight)/control group tumor average weight × 100%. The organ index of the liver and spleen was determined as organ weight (g)/body weight (g).

The tumor tissue was fixed with paraformaldehyde for 48 h, embedded in pure paraffin, and cut into sections of 4∼5 μm. Tissue sections were attached to slides, dewaxed in xylene, rehydrated in ethanol, and stained with hematoxylin and Eosin. The morphology of HE-stained tumor tissue was observed under an optical microscope (Nikon Eclipse E100, Japan).

Tumor tissue sections were dewaxed with xylene, then hydrated, and antigen repair was performed using citric acid (PH6.0) antigen repair solution (Servicebio, Wuhan, China). After blocking endogenous peroxidase, incubated with primary antibody at 4°C overnight, and then the tissue sections were incubated with secondary antibody at room temperature for 50 min. Finally, after DAB staining and neutral gum sealing, microscopic examination and image acquisition were performed. Quantitative analysis of IHC data was detected by Image-Pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD).

All experimental results were expressed as the mean ± standard deviation (SD) of triplicate samples. Statistical analysis was performed using IBM SPSS Statistics 26.0 (IBM, Armonk, United States) and GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, United States). One-way analysis of variance and Dunnett multiple comparison test were used to determine the differences between the average values of different groups. Significance was determined at values *p < 0.05, **p < 0.01, ***p < 0.001.

The effect of MG (0–640 μM) on the proliferation of BEL-7402 and LO2 cells was quantified using the MTT assay, wherein 5-FU (50 μM) was used as the positive control. As shown in Figure 1A, MG inhibited BEL-7402 cell proliferation in a time- and concentration-dependent manner. Moreover, MG showed minimal toxicity in human normal hepatocyte LO2 cells (Figure 1B). The toxicity of MG in normal human liver cells, LO2 was less than 1/11th of that in the human liver cancer cell line, BEL-7402, and less than 1/7th of that of the positive drug 5-FU. These findings (Figures 1A,B) suggest that MG inhibits the proliferation of BEL-7402 cells in vitro, and it is far less toxic. Colony formation assay also showed that MG significantly inhibited the growth of BEL-7402 cells compared to that of the control cells (Figure 1C).

Cell migration and invasion are closely associated with tumor metastasis. As shown in Figures 2A,B, after 24 and 48 h of MG administration, scratch assay revealed that BEL-7402 cells in the control group healed gradually, and cell migration activity in the drug-treated groups reduced with an increase in MG concentration compared to that in control cells. Invasion ability was measured using the transwell cell invasion assay, wherein transwell pores were coated with Matrigel. The number of invaded cells in the MG-treated group was significantly lower than that in the control group. At 80 and 160 μM drug concentrations, the number of invaded cells after 24 h of treatment was significantly higher than that after 48 h of treatment (Figures 2C,D). These findings indicate that MG inhibits the migration and invasion of BEL-7402 cells in a time- and dose-dependent manner in vitro.

To explore the underlying mechanism of MG-mediated inhibition of migration and invasion of HCC cells, BEL-7402 cells were treated with different concentrations of MG (40, 80, and 160 μM) for 48 h, and the protein expression levels of AMPK, NF-κB, p-NF-κB, MMP9, MMP2, TIMP-2, E-cadherin, and vimentin were quantified (Figure 3). MG treatment decreased the expression of AMPK, NF-κB, and p-NF-κB in a dose-dependent manner compared to that in the control group (Figure 3A). MMPs are essential for migration and invasion of cancer cells and degradation of the ECM (Scheau et al., 2019). Thus, we examined the inhibitory effect of MG on MMPs. Our results showed that MG significantly reduced the expression of MMP2 and MMP9 and increased the expression of TIMP-2, which is a negative regulator of cell matrix degradation, compared to that in the control group (Figure 3B). EMT is considered to be a critical event in cancer metastasis. EMT involves the downregulation of epithelial adhesion molecules, such as E-cadherin and cytokeratin, and upregulation of mesenchymal markers, such as vimentin and α-smooth muscle actin (α-SMA) (Brabletz et al., 2021). Our results showed that MG significantly increased E-cadherin and decreased vimentin protein expression at medium and high doses compared to that in the control group, significantly inhibiting EMT (Figure 3C).

The safety and efficacy of MG in vivo were investigated in a xenograft model. The average tumor volumes and weights of the MG experimental group and the 5-FU positive group were significantly lower than those of the control group (Figures 4A–D). The tumor inhibition rates of 40, 80, 160 mg/kg/d of MG groups, 5-FU group, and combination group were found to be 49.94, 52.66, 55.92, 70.18, and 74.75%, respectively. Interestingly, the combination of MG and 5-FU had the strongest therapeutic effect, indicating that MG potentially promotes inhibition of tumor proliferation by 5-FU. Nonetheless, the mechanism requires further verification. MG and 5-FU had no effect on liver and spleen indices (Figures 4F,G). In the later stage of tumor induction and drug administration, it was observed that the body weight of nude mice in the MG 40, 80, and 160 mg/kg/d dose groups increased over time and was higher than that in the 5-FU and combined medication groups (Figure 4E). However, there was no significant physiological change in nude mice during the treatment, indicating that MG was well tolerated, relatively non-toxic, and safe for mice.

Pathological examination of xenografts from different groups was performed using HE staining. In the control group, we found large tumors with closely arranged cells, which were large in size with evident nuclear diversity and deep staining. After drug treatment, necrosis and apoptotic cells in tumor tissue gradually increased in size, cells were arranged loosely, and interstitial spaces increased. Furthermore, there was an increase in nuclear fragmentation, volume shrinkage, and chromatin condensation in the nucleus (Figures 5A,B). IHC was used to detect the expression of NF-κB, MMP9, MMP2, and TIMP-2, which are the four key targets for tumor migration and invasion. Compared with that in the control group, the expression of NF-κB, MMP9, and MMP2 in the 80 mg/kg/d MG group was significantly decreased, and the expression of TIMP-2 was markedly increased. The effects of 5-FU on these proteins were similar to those in the 80 mg/kg/d MG group (Figures 5C–G).