Human hepatocellular carcinoma cells MHCC97-L and HCCLM3 (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.), and incubated at 37°C in an atmosphere containing 5% CO₂. Media were supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). EA was purchased from Chengdu Must Bio-Technology Co., Ltd. and was dissolved in DMSO before being used to treat the hepatocellular carcinoma cell lines MHCC97-L and HCCLM3 at different concentrations (7, 14 and 28 µM) at 37°C for 24, 48 or 72 h, according to the subsequent experiment. The following inhibitors were used to treat the MHCC97-L and HCCLM3 cell lines for 30 min prior to EA treatment at 37°C: Z-VAD-FMK (20 µM; MedChemExpress LLC), necrostatin-1 (30 µM; MedChemExpress LLC), 3-methyladenine (3-MA; 2 mM; MedChemExpress LLC), N-acetylcysteine (NAC; 3 mM; MilliporeSigma) and PD98059 (20 µM; MedChemExpress LLC). DMSO was used to treat the control group. All experiments were performed independently and in triplicate.

Hepatocellular carcinoma cells (4×10³ cells/well) were seeded into six-well cell culture plates and incubated for 48 h after EA (7, 14 and 28 µM) and DMSO treatment. The cells were observed under a light inverted microscope (Leica Microsystems GmbH). Cell numbers were analyzed using Image-Pro Plus 7.0 software (Media Cybernetics, Inc.). The proliferation rate was calculated as follows: 100× (mean number of cells in the experimental group/mean number of cells in control group).

The CCK8 (Dojindo Laboratories, Inc.) was used to determine the viability of MHCC97-L and HCCLM3 cells. Cells were seeded into 96-well culture plates at a density of 5,000 cells/well. EA was then added to the drug administration group at a final concentration of 7, 14 and 28 µM, and the cells were incubated for 24, 48 and 72 h at 37°C. CCK8 solution (10 µl) was then added to each well and the cells were further incubated at 37° for 4 h. The OD value of the cells was measured at 450 nm using a microplate reader.

Cell proliferation was measured by BrdU staining. A total of 1×10⁴ cells were seeded in 24-well plates, followed by the addition of DMEM containing DMSO or EA (28 µM), and were incubated at 37°C for 2 days. Subsequently, the cells were incubated with 10 µg/ml BrdU (MilliporeSigma) for 2 h at 37°C and fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were then treated with 2 M HCl at 37°C for 30 min and blocked with 10% goat serum (OriGene Technologies, Inc.) containing 0.3% Triton X-100 for 1 h at room temperature. The cells were then incubated with BrdU primary antibody (1:1,000; cat. no. ab6326; Abcam) at 4°C overnight and with an Alexa Fluor™ 594-conjugated goat anti-rat IgG secondary antibody (1:1,000; cat. no. A-11007; Thermo Fisher Scientific, Inc.) for 2 h at room temperature. The cells were finally stained with DAPI for 15 min at room temperature and observed under a fluorescence microscope. BrdU-positive cells in three random fields were counted. The cells were counted using Photoshop CC 2018 software (Adobe Systems, Inc.).

The effect of EA on the colony-forming ability of hepatocellular carcinoma cells was determined using the soft agar assay. Briefly, 1.5 ml 2X RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) containing 0.6% agarose (MilliporeSigma) was added to 6-well plates as base agar at 37°C for 1 h. A total of 10³ cells and 28 µM EA were then added to the top of the base agar and incubated at 37°C for 21 days. The hepatocellular carcinoma cells were finally stained with MTT for 30 min at 37°C and were images were captured using a digital camera.

Hepatocellular carcinoma cells were seeded in a 6-well plate and incubated until they reached 90% confluence. The cells were then scratched using a 100-µl sterile pipette tip to form a linear wound, followed by the addition of serum-free DMEM containing 7, 14 or 28 µM EA or DMSO. Cell migration across the wounded area was observed at 6, 24 and 48 h and the extent of wound closure was measured at the indicated time points (14). The cells were observed under a light inverted microscope (Leica Microsystems GmbH). Cell migration was analyzed using Image-Pro Plus 7.0 software. The wound closure rate was calculated as follows: Wound closure (%)=100× [scratch width at 0 h-scratch width at 6, 24 or 48 h)/(scratch width at 0 h)].

The migratory potential of hepatocellular carcinoma cells was measured using Transwell chambers (pore size, 8 µm; Corning, Inc.). Briefly, the hepatocellular carcinoma cells (2×10⁵) were suspended in DMEM containing 1% FBS and were seeded into the upper chamber. DMEM (500 µl) containing 20% FBS was then added to the lower chamber to act as a chemoattractant. The cells were incubated for 48 h with DMEM at 37°C containing 7, 14 or 28 µM EA, or DMSO. Cells on the lower surface of the membrane were stained with 0.5% crystal violet for 15 min at room temperature. The cells were observed under an light inverted microscope (Leica Microsystems GmbH). Images of the stained cells were randomly captured and the cells were counted using the Image-Pro Plus 7.0 software. The migration rate was calculated as follows: 100× (mean number of migrating cells in the experimental group/mean number of migrating cells in control group).

Western blotting was performed as previously described (15). The cells were treated with EA (7, 14 and 28 µM) for 24 h. The treated cells were harvested and lysed on ice using RIPA lysis buffer (Beyotime Institute of Biotechnology) containing protease and phosphatase inhibitors (Roche Diagnostics). The protein concentration of the cell lysate was measured using the BCA protein assay kit (Beyotime Institute of Biotechnology). The cell lysate was then denatured at 100°C for 5 min. Subsequently, 50 µg protein extracts were subjected to SDS-PAGE on 10 and 12% gels and were transferred onto a polyvinylidene difluoride membrane. The membrane was blocked in 5% bovine serum albumin (MilliporeSigma) at room temperature for 1 h, and was then incubated overnight at 4°C with the following primary anti-bodies: E-cadherin (1:1,000; cat. no. 14472), Vimentin (1:1,000; cat. no. 5741T), cleaved-PARP (1:1,000; cat. no. 5625T), cleaved-caspase-3 (1:1,000; cat. no. 9661), autophagy-related 5 (Atg5; 1:1,000; cat. no. 12994T), p62 (1:1,000; cat. no. 8025T), MAPK family sampler kit (1:1,000; cat. no. 9926T), p-ERK (1:1,000; cat. no. 4370T), p-JNK (1:1,000; cat. no. 4668T), p-p38 (1:1,000; cat. no. 4511T), LC3B (1:1,000; cat. no. 12741), Cyclin D1 (1:1,000; cat. no. 55506), fibronectin (1:1,000; cat. no. 26836), CDK4 (1:1,000; cat. no. 12790), PARP (1:1,000; cat. no. 9542), caspase-3 (1:1,000; cat. no. 9662) (all Cell Signaling Technology, Inc.), CDK2 (1:500; cat. no. sc-6248), Cyclin E1 (1:500; cat. no. sc-247) (both Santa Cruz Biotechnology, Inc.), RIP1 (1:1,000; cat. no. CY6582), p-MLKL (1:1,000; cat. no. CY7146), MLKL (1:1,000; cat. no. CY5493) (all Shanghai Abways Biotechnology Co., Ltd.), N-cadherin (1:1,000; cat. no. AP71171), ZEB1 (1:1,000; cat. no. AM1878A) (both from Abcepta Biotech Ltd. Co.) and β-actin (1:1,000; cat. no. TA-09; OriGene Technologies, Inc.). The membrane was subsequently incubated with horseradish peroxidase-conjugated secondary antibodies [goat anti-mouse IgG (cat. no. A0216) and goat anti-rabbit IgG (cat. no. A0208); 1:10,000; Beyotime Institute of Biotechnology] at room temperature for 1 h. The protein bands were finally visualized using the Clarity Western ECL Blotting Substrate (cat. no. 1705061; Bio-Rad Laboratories, Inc.) and images were captured using a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.). The integrated optical density of the protein bands was measured using the Bio-Rad Image Lab Software 5.2.1 (Bio-Rad Laboratories, Inc.), with β-actin employed as the loading control.

Hepatocellular carcinoma cells were incubated for 48 h after EA (14 and 28 µM) at 37°C and DMSO treatment. The treated cells at 70-80% confluence were fixed with 75% ethanol at 4°C and then incubated in 500 µl PBS containing PI and RNase A (9:1) at 37°C for 60 min (Cell Cycle Kit; Nanjing KeyGen BioTech Co., Ltd.). The cell samples were analyzed using DxFLEX flow cytometry (Beckman Coulter, Inc.). The cell percentage in each cycle was finally evaluated using Modfit-LT V 3.0 software (Verity Software House).

Hepatocellular carcinoma cells were cultured for 48 h after EA (7, 14 and 28 µM) and DMSO treatment. The cells were then collected, washed with cold PBS and suspended in 200 µl binding buffer (Annexin V-FITC/PI apoptosis assay kit; Neobioscience Technology Co., Ltd.) at a density of 5×10⁵/ml. Subsequently, cells in binding buffer (195 µl) were incubated with 5 µl Annexin V-FITC at room temperature in the dark for 10 min. The cells were then washed with PBS, suspended in binding buffer, and mixed with 10 µl PI. The mixture was gently mixed and analyzed by DxFLEX flow cytometry. The apoptotic rate of the hepatocellular carcinoma cells was finally determined using CytExpert 2.0 software (Beckman Coulter, Inc.).

Hepatocellular carcinoma cells were incubated for 48 h after EA (28 µM) and DMSO treatment. The cells at a density of 10⁶/ml were then collected into a 1.5 ml Eppendorf tube containing trypsin and centrifuged at 100 × g for 5 min at room temperature. The supernatant was collected, and the cells were fixed with 1 ml 2.5% glutaraldehyde fixing solution for 1 h at 4°C and treated with 1% OsO₄ in PBS for 1 h at 4°C The cells were then dehydrated in ethanol, embedded in epoxy resin and sliced into 90-nm sections. The sections were stained with uranyl acetate for 5 min and lead citrate for 5 min at 37°. Ultrathin sections of the cells were examined under a JEM1200EX transmission electron microscope (Jeol Ltd.).

Hepatocellular carcinoma cells were incubated for 48 h after EA (14 and 28 µM) and DMSO treatment. The cells (4×10³ cells/well) were seeded into six-well plates, then collected, suspended in serum-free DMEM containing 10 µM DCFH-DA (Reactive Oxygen Species Assay Kit; Beyotime Institute of Biotechnology) and incubated at 37°C for 20 min. The cells were subsequently washed three times with serum-free cell medium and analyzed by DxFLEX flow cytometry. The ROS content in the hepatocellular carcinoma cells was finally analyzed using CytExpert 2.0 software.

HCCLM3 cells were harvested after 48 h of treatment with DMSO and EA (14 and 28 µM) at 5% CO₂ and 37°C for total RNA isolation using TRNzol Universal Total RNA extraction reagent (cat. no. DP424, Tiangen Biotech Co., Ltd.). RNA samples were submitted to Shanghai Personalbio Technology Co., Ltd. for transcriptome sequencing and analysis. The concentration, quality and integrity of RNA were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). Integrity was assessed by an Agilent 2100 Bioanalyzer using the RNA 6000 Nano kit (cat. no. 5067-1511) (both Agilent Technologies Inc.). Subsequently, 3 µg RNA was used as the input material for RNA sample preparation. The experiment was performed according to manufacturer's protocol of NEBNext Ultra II RNA Library Prep Kit for Illumina (cat. no. E7775; New England Biolabs, Inc.). Sequencing libraries were generated according to the following steps. Firstly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer (Illumina, Inc.). First strand cDNA was synthesized using random oligonucleotides and Super Script II. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3' ends of the DNA fragments, Illumina PE adapter oligonucleotides (Illumina, Inc.) were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Inc.).

DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail (Illumina, Inc.) in a 15-cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent High Sensitivity DNA assay on the 2100 Bioanalyzer system (both Agilent Technologies, Inc.). The sequencing library was then sequenced on the NovaSeq 6000 platform (Illumina, Inc.) by Shanghai Personal Technology Co. Ltd. To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system. The library was uniformly diluted to 2 nM and PE150 mode sequencing was performed on the Illumina sequencer.

The original off-machine data were filtered for the transcriptome analysis. The high-quality sequences obtained were aligned with the human reference genome (Homo_sapiens. GRCh38.dna.primary_assembly.fa). The expression levels of the genes were calculated based on the comparison results, followed by expression difference analysis between the EA (24 µM) and control group, enrichment analysis and cluster analysis between EA and Control groups. Gene read counts were estimated with HTSeq (0.9.1) statistics (16). FPKM was used to standardize the expression. The differential expression of genes was analyzed using DESeq 1.30.0 (Bioconductor) (16,17). The screening conditions were as follows: |log2FoldChange|>1 and significant P<0.05. The R language heatmap (1.0.8) software package was used to perform a bi-directional clustering analysis of all different genes in the samples (16). The heatmap was developed according to the expression levels of the same gene in different samples and the expression patterns of different genes in the same sample. The Euclidean method was used to calculate the distance and the complete linkage method was performed for clustering. ClusterProfiler (3.4.4) software was used to carry out the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed genes, focusing on the pathways with P<0.05 (18-20).

A total of 36 female nude mice (BALB/c; weight 18-20 g; age, 4 weeks) were purchased from Cavensbiogle and housed in a specific pathogen-free room that was maintained at a constant temperature (20-26°C) and humidity (40-70%). The mice were maintained under a 12-h light/dark cycle with free access to food and water. The animals were divided into three groups and the number of mice per group was six; the number of mice in the MHCC97-L group was 18 and the number of mice in the HCCLM3 group was also 18. MHCC97-L and HCCLM3 cells (1×10⁶ cells in 200 µl PBS) were subcutaneously injected into the nude mice. The mice were then intraperitoneally injected with EA (30 or 60 mg/kg) and saline solution (Control) once per day for 3 weeks. The body weight of the mice was monitored, and the tumor size was measured to calculate the tumor volume, as follows: Volume=4π/3 × R³, where R=L + W/2; R refers to the radius of tumor, L refers to the longest diameter of the tumor mass and W to the longest transverse diameter perpendicular to the longest diameter. Volume was calculated from day 12 to 26 (once every 2 days). The mice were euthanized using CO₂ (30% of the volume of the euthanasia chamber was replaced with CO₂ every minute) on day 26 (the maximum tumor volume of the MHCC97-L group was 810.41 mm³ and the maximum tumor volume of the HCCLM3 group was 960.66 mm³), and the tumors were subsequently excised and weighted.

Data were analyzed using GraphPad Prism 5.01 software (GraphPad Software, Inc.) and were presented as the mean ± SD. All experiments were repeated at least three times. Two groups were compared using an unpaired two-tailed Student's t-test. One-way ANOVA followed by Tukey's test was used to assess and compare the mean differences among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Hepatocellular carcinoma is characterized by rapid development and high mortality (21). Effective inhibition of tumor growth is therefore of great importance. The concentration of EA used in the present study was chosen based on relevant studies of EL extract and the IC₅₀ of EA in MHCC97-L and HCCLM3 cells (Fig. S2B) (22,23). When the concentration of EA was ~10 µM, it could serve a significant inhibitory effect on hepatocellular carcinoma cell lines; therefore, the concentrations of EA used in the present study were selected. In the present study, MHCC97-L and HCCLM3 cells were treated with different concentrations of EA (7, 14 and 28 µM) for 48 h to investigate the anti-proliferative effects of EA on human hepatocellular carcinoma cells. DMSO was used as the control. The viability of hepatocellular carcinoma cells was examined through light morphological analysis, CCK8 assay, BrdU staining and soft agar assay. Microscopic examinations revealed that EA altered their morphology in a dose-dependent manner (Fig. 1A). Decreased cell number, cell shrinkage, dispersed cell growth and pleomorphic cell morphology were observed following EA treatment. Images of the cells were captured and the number of cells was statistically analyzed to obtain the proliferation rate. The proliferation rate was measured by comparing the proliferation of the DMSO group with that of the EA group and the proliferation rate was reduced following EA treatment (Fig. 1B). Notably, there was a significant decrease in the viability of the two cell lines treated with EA after 24, 48 and 72 h compared with that in the DMSO group (Fig. 1C). DNA synthesis in cells treated with EA was also reduced (Fig. 1D and E). The soft agar assay further revealed smaller and fewer colonies following EA treatment (28 µM EA for 48 h) compared with the colonies in the DMSO group (Fig. 1F and G). These results suggested that EA markedly inhibited the viability and proliferation of human hepatocellular carcinoma cells.

Surgical resection remains the main treatment method for hepatocellular carcinoma and it significantly reduces the mortality of patients with hepatocellular carcinoma; however, the postoperative recurrence rate remains high at >70% (24). Therefore, the present study assessed whether EA affected cell motility using the wound-healing assay. Notably, there was a significant decrease in cell migration following EA treatment for 24 and 48 h compared with that in the DMSO group (Fig. 2A and B). The results of Transwell assays were similar to the results of the wound-healing assay (Fig. 2C and D). E-cadherin, Vimentin, N-cadherin, fibronectin and ZEB1 are major canonical markers of the epithelial-mesenchymal transition (EMT), which serve crucial roles in the tumor migration process (25). In the present study, E-cadherin, N-cadherin, fibronectin and ZEB1 were significantly upregulated, whereas Vimentin was significantly downregulated following EA treatment in a dose-dependent manner (Figs. 2E and F, and S1A and B). These results suggested that EA treatment reversed the EMT of human hepatocellular carcinoma cells, thus strongly inhibiting their migration.

The proliferation of tumor cells is determined by cell cycle progression (26). Antitumor drugs can be divided into cell cycle-specific and non-specific drugs (27). In the present study, the effect of EA on the cell cycle was examined using flow cytometry. The percentage of MHCC97-L and HCCLM3 cells in the G₁ phase was significantly increased following treatment with 14 and 28 µM EA for 48 h compared with the control cells (Fig. 3A and B). Notably, the expression levels of CDK2, CDK4, cyclin E1 and cyclin D1, the key regulators for G₁ phase transition (28), were downregulated following treatment with 14 and 28 µM EA (Figs. 3C and D, and S1C and D). These results demonstrated that EA induced G₁-phase arrest by affecting the expression of cell cycle regulatory proteins.

Apoptosis and necrosis are the major cell death mechanisms (29), and numerous antitumor drugs exert their effects by inducing apoptosis or necrosis (30,31). The present study therefore investigated whether EA treatment caused apoptosis and necrosis of hepatocellular carcinoma cells by detecting the expression levels of apoptosis- and necrosis-related proteins using western blot analysis following treatment of the cells with 7, 14 and 28 µM EA for 48 h. Notably, EA treatment did not affect the expression levels of cleaved-PARP and cleaved-caspase-3, which are apoptosis-related proteins, nor those of RIP1 and p-MLKL, which are necrosis-related proteins (Fig. 4A and B). The Annexin V-FITC/PI apoptosis assay conducted using flow cytometry further revealed that EA did not induce early or late apoptosis (Fig. 4C and D). Furthermore, treatment with the apoptosis inhibitor (Z-VAD-FMK) and necrosis inhibitor (necrostatin-1) did not restore the EA-induced decrease in cell viability (Fig. 4E). These results suggested that EA-induced inhibition of human hepatocellular carcinoma cell viability was independent of apoptotic and necroptotic factors.

Autophagy is a classic type of programmed cell death that participates in the functional modulation of cancer cells by affecting Atg proteins (32). The present study investigated whether EA served an anti-liver cancer role by inducing autophagy. Notably, EA upregulated the expression levels of LC3 II/I and Atg5, but downregulated p62/SQSTM1 expression, compared with those in the DMSO group (Fig. 5A-F). LC3 II/I, Atg5 and p62/SQSTM1 are autophagy biomarkers. Autophagosomes, which are two-layered structures containing cytoplasmic components, were formed following treatment with 28 µM EA (Fig. 5G). ROS regulate the autophagy pathway in cancer cells (33-35). Notably, excessively high levels of ROS can induce cell death through oxidative damage (36). In the present study, EA strongly induced ROS production compared with in DMSO cells (Fig. 5H and I). Treatment with the pharmacological inhibitor of autophagy 3-MA and anti-ROS reagent NAC efficiently reversed the EA-induced inhibition of cell viability and migration (Fig. 5J and K). These results suggested that autophagy and ROS activation potentially contributed to human hepatocellular carcinoma cell death and defects in migration induced by EA.

Abnormal activation of the MAPK signaling pathway is closely related to the occurrence, development and metastasis of hepatocellular carcinoma (37). In the present study, transcriptome analysis, which was conducted to investigate the mechanism underlying EA-induced cell death and migration, revealed that gene expression levels were significantly altered after EA treatment compared with in the control group (Fig. 6A). According to the KEGG pathway enrichment analysis results of differentially expressed genes, the top 10 pathways with the minimum P-value or the most significant enrichment were selected (Fig. 6B). The present study focused on the 'MAPK signaling pathway'. Further validation of the transcriptome sequencing data using western blotting revealed that EA significantly upregulated p-ERK expression, but did not affect p-p38 and p-JNK expression, in MHCC97-L and HCCLM3 cells compared with in the DMSO group (Figs. 6C and S2A). Furthermore, the decreased cell viability and migration induced by EA were reversed by ERK inactivation using PD98059 (Fig. 6E-G). These results suggested that ERK signaling participated in EA-induced cell death and migration inhibition.

A ROS scavenger (NAC) and ERK-specific inhibitor (PD98059) were used to investigate the interplay between ROS and ERK in EA-induced autophagy using western blotting. Pretreatment with NAC significantly reduced the activation of p-ERK and LC3 II/I induced by EA in both MHCC97-L and HCCLM3 cells, indicating that ROS was an upstream signal of EA and induced ERK activation and autophagy (Fig. 7A-D). Notably, the ERK-specific inhibitor PD98059 also suppressed the EA-induced increased expression of p-ERK and LC3 II/I in both MHCC97-L and HCCLM3 cells (Fig. 7E-H). These data suggested that EA induced autophagy by activating ROS production and ERK signaling.

In vivo assays using nude mice were further performed to determine whether EA inhibited the growth of hepatocellular carcinoma. Nude mice were subcutaneously inoculated with 1×10⁶ hepatocellular carcinoma cells. The mice were subsequently intraperitoneally injected with EA or DMSO (control) daily for 21 days, followed by an assessment of tumor growth following cessation of treatment. Mice treated with EA exhibited a smaller tumor volume and weight than mice in the control group (Fig. 8A-C), thus indicating that EA markedly inhibited tumor growth in vivo.