HepG2 and HepG2/5-FU cells were purchased from the Cell Conservation Center of Xiangya Medicine College, Hunan University (Hunan, China) and they were bought from American Type Culture Collection (ATCC, cat. no. ATCC^® HB-8065). The cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (cat. no. 16000-044, Gibco; Thermo Fisher Scientific, Inc.). The cells were seeded in 6-, 12- or 24-well plates, diluted to the final concentration of 5.0×10⁵ cells/ml and incubated at 37°C in a humidified incubator with an atmosphere of 5% CO₂ under aseptic conditions.

KGM was purchased from Career Henan Chemical Co. (cat. no. 37220-17-0; http://www.chemicalbook.com/ProductDetail_EN_450429.htm), and its purity was 99%; 5-FU was purchased from Sigma Aldrich; Merck KGaA (cat. no. F6627-1G). 5-FU and KGM were diluted in DMSO and water, respectively.

Cell viability or proliferation was determined using an MTT assay. The cells (HepG2 or HepG2/5-FU) were seeded in 96-well plates at a density of 1.0×10⁴ cells/well in 0.1 ml RPMI-1640 medium and were exposed to increasing concentrations of 5-FU (0.001, 0.005, 0.020, 0.080, 0.320, 1.280, 2.560, 5, 10, 20, 40, 80 or 160 µM) or KGM (0, 2, 4, 8, 10, 100 or 1,000 µg/ml) for 24 h.

MTT (cat. no. 57360-69-7; Sigma-Aldrich; Merck KGaA) was dissolved in DMSO to 5 mg/ml; the cells were incubated with MTT for 4 h at 37°C. The absorbance of the samples was measured using a microtiter plate reader at 490 nm.

To investigate the reversal effects of KGM on the viability of HepG2/5-FU cells, the cells were preincubated with KGM (2 or 6 µg/ml) and 5-FU (0.5 µM) for 24 h at 37°C. The absorbance of the samples at 490 nm was determined by MTT assay as described above.

Apoptosis was determined using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. HepG2/5-FU cells were seeded in 96-well plates at a density of 5.0×10⁵ cells/well. The cells were treated with 2 or 6 µg/ml KGM for 24 h at 37°C in the presence of 5-FU. Subsequently, the cells were stained using an Annexin V-FITC/PI Apoptosis Detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The cells were analyzed using a BD FACScan flow cytometer (Becton, Dickinson and Company) and CellQuest acquisition and analysis software v.3.0 (Becton, Dickinson and Company). Early, late or early + late apoptosis was assessed and the early apoptotic cells were stained by Annexin V-FITC and the late apoptotic cells were stained by both Annexin V-FITC and PI.

The cells (HepG2 or HepG2/5-FU) were lysed in RIPA Buffer (Beyotime Institute of Biotechnology), and protein concentration was measured by bicinchoninic acid assay (Beyotime Institute of Biotechnology). Total protein (50 µg) was resolved using 10% SDS-PAGE, electro-transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime Institute of Biotechnology) and blocked with 5% non-fat dry milk in Tris-buffered saline, pH 7.5 for 30 min at the room temperature. The PVDF membranes were incubated with primary antibodies against MDR1 (1:1,000; cat. no. 13978; Cell Signaling Technology Inc.); anti-P-gp (1:1,000; cat. no. ab170904; Abcam); Cyclin A1 (1:1,000; cat. no. 4656; Cell Signaling Technology Inc.); anti-cyclin B1 (1:1,000; cat. no. 4138; Cell Signaling Technology Inc.); CDK2 (1:1,000; cat. no. 2546; Cell Signaling Technology Inc.); Bcl-2 (1:1,000 dilution; cat. no. 4223; Cell Signaling Technology Inc.); Bax (1:1,000 dilution; cat. no. 14796; Cell Signaling Technology Inc.); GAPDH (1:1,000; cat. no. MB001; Bioworld, Technology Inc.); cleaved caspase-3 (1:1,000; cat. no. P42574; Bioworld Technology Inc.); p53 (1:1,000; cat. no. 9282; Cell Signaling Technology Inc.); E-cadherin (1:1,000; cat. no. 14472; Cell Signaling Technology Inc.); N-cadherin (1:1,000; cat. no. 13116; Cell Signaling Technology Inc.); AKT (1:1,000; cat. no. 4685; Cell Signaling Technology Inc.) and p-AKT (1:1,000 dilution; cat. no. 9611; Cell Signaling Technology Inc.) for 5 h at room temperature and the membranes were washed with PBST (Tween-20-containing phosphate-buffered saline) and incubated with horseradish peroxidase (HRP)-conjugated mouse or rabbit IgG secondary antibodies anti-HRP IgG (1:5,000; cat. no. 7074; Cell Signaling Technology Inc.) and anti-HRP IgG (1:5,000; cat. no. 7076; Cell Signaling Technology Inc.) for 1 h at 37°C. Protein bands were detected by an enhanced chemiluminescence kit (Invitrogen; Thermo Fisher Scientific, Inc.) and quantified using ImageJ v.1.8.0 software (National Institutes of Health).

Total RNA was extracted from the cells (HepG2 or HepG2/5-FU) with TRIzol^® reagent (Sangon Biotech Co., Ltd.,), and cDNA synthesis was performed using the PrimeScript II 1^st Strand cDNA Synthesis kit (Takara Bio, Inc.) according to the manufacturer's instructions. QPCR was performed with a SYBR^® Green PCR system (Takara Bio, Inc.) in an ABI 7500 thermal cycler (Thermo Fisher Scientific, Inc.), and the thermocycling conditions were as follows: 94°C for 5 min; followed by 35 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for 15 sec. The primers used were as follows: GAPDH forward, 5′-GCAGTGGCAAAGTGGAGATT-3′ and reverse, 5′-TGAAGTCGCAGGAGACAACC-3′; MDR1 forward, 5′-TCACTTCAGTTACCCATCTCG-3′ and reverse, 5′-CACCAATGATTTCCCGTAG-3′; P-gp forward, 5′-ACTTGCAAGGGGACCAGAGA-3′ and reverse, 5′-CCTTCAAGATCCATTCCGACC-3′; cyclin A forward, 5′-TCCATGTCAGTGCTGAGAGGC-3′ and reverse, 5′-GAAGGTCCATGAGACAAGGC-3′; cyclin B1 forward, 5′-ATGCAGCACCTGGCTAAGAA-3′ and reverse, 5′-TTACACCTTTGCCACAGCCT-3′; CDK2 forward, 5′-CTTTGCTGAGATGGTGACTCG-3′ and reverse, 5′-TCATCCAGGGGAGGTACAACT-3′; and caspase-3 forward, 5′-TGCATACTCCACAGCACCTG-3′ and reverse, 5′-TCTGTTGCCACCTTTCGGTT-3′. RT-qPCR for each sample was performed in duplicate. The fold-changes were calculated by relative quantification (2^−ΔΔCq) (21). GAPDH was used as an internal control.

BALB/c male nude mice (4-week-old) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China) and divided into 2 mice/group. All animal experiments were performed in accordance with the relevant guidelines of the Institutional Animal Care and Use Committee of Nanjing Medical University (approval number, SYXK 2015-0015). HepG2/5-FU cells (150 µl, 4.0×10⁶ cells) were injected subcutaneously into the right flank of athymic mice. The mice were housed in an isolated, clean, air-conditioned room at 24–26°C and a relative humidity of ~50% with a 12/12-h light/dark cycle and they had free access to food and water. The tumors were inspected every other day. Drug treatment was started when the tumor volume reached 40–50 mm³ (22). The mice were randomly divided into two groups 5-FU (2 mg/kg) alone and 5-FU (2 mg/kg) plus KGM (20 mg/kg) groups with two mice in each group. The drugs were administered every 2 days i.p. The tumor growth was determined every other day, and the tumor volume was calculated using the following formula: Volume = (longest diameter × shortest diameter2) / 2 (22). On day 13, the tumor-bearing mice were sacrificed using the cervical dislocation method and the tumors were weighed.

The data are presented as the mean ± standard deviation. All assays were performed as three independent experiments. Data were analyzed using Student's t-test or one-way ANOVA followed by Tukey's honestly significant difference test using SPSS 17.0 software (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.

MTT assays were performed to measure the effects of 5-FU and KGM on the viability of liver cancer cells. The results demonstrated that 5-FU exhibited cytotoxic activity on the HepG2 and HepG2/5-FU cells with 50% inhibitory concentration (IC₅₀) values of 1.03 and 60.02 µM, respectively (Fig. 1A). By contrast, the IC₅₀ of KGM was 1,500.41 µg/ml in HepG2 cells (Fig. 1B). Thus, 5-FU exhibited different inhibition in drug-sensitive and MDR cancer cells. In addition, 5-FU exhibited no effects on the HepG2/5-FU cell viability at ≤0.6 µM (Fig. 1A), and KGM exhibited no cytotoxic activity on HepG2/5-FU cells at ≤8 µg/ml (Fig. 1B). Thus, to retain the 5-FU-resistant properties of HepG2/5-FU, the cells were cultured in medium containing 0.2 µM 5-FU.

To investigate the reversal effects of KGM on HepG2/5-FU cells, the cells were incubated with 0.5 µM 5-FU and/or 2 or 6 µg/ml KGM for 24 h. The results demonstrated that 0.5 µM 5-FU exhibited no cytotoxic activity on HepG2/5-FU cells (Fig. 1A), and that 2 or 6 µg/ml KGM also exhibited no cytotoxicity to this drug-resistant cell line (Figs. 1B and 2A). However, in the presence of 0.5 µM 5-FU, 2 and 6 µg/ml KGM significantly decreased HepG2/5-FU cell viability (Figs. 1B and 2A). In addition, the mRNA and protein levels of P-gp and MDR were significantly downregulated by KGM in the presence of 0.5 µM 5-FU compared with 5-FU or KGM treatment alone (Fig. 2B-D).

To investigate the effects of KGM on HepG2/5-FU cell proliferation, the expression levels of the cell cycle-related genes cyclin A, cyclin B1 and CDK2 were determined. The results demonstrated that no significant differences were observed between the protein or mRNA levels of cyclin A, cyclin B1 and CDK2 in HepG2/5-FU cells following incubation with 0.5 µM 5-FU or 2 µg/ml KGM alone and the control groups. By contrast, increasing concentrations of KGM in the presence of 0.5 µM 5-FU significantly reduced the protein and mRNA levels of cyclin A, cyclin B1 and CDK2 in HepG2/5-FU compared KGM treatment alone (Fig. 3A-D).

The expression of the proapoptotic gene caspase-3 was upregulated in HepG2/5-FU cells after KGM treatment in the presence of 0.5 µM 5-FU compared with 5-FU or KGM treatment alone (Fig. 3E). Additionally, the protein levels of the antiapoptotic gene BCL-2 level were reduced, whereas the protein levels of the proapoptotic genes Bax and caspase-3 were increased after KGM treatment in the presence of 0.5 µM 5-FU in HepG2/5-FU cells compared with 5-FU or KGM treatment alone (Fig. 3F).

KGM also significantly promoted HepG2/5-FU apoptosis; KGM increased the apoptotic rate (1.85±0.21) in cells treated with 0.5 µM 5-FU to 5.06±0.62 and 26.91±1.73% in cells treated with 0.5 µM 5-FU and 2 or 6 µg/ml KGM, respectively (Fig. 3G and H).

To explore the potential mechanisms of KGM in reversing effects of MDR, p53 expression and AKT phosphorylation in KGM-incubated HepG2/5-FU cells was investigated. The results demonstrated that KGM significantly upregulated p53 and E-cadherin and decreased phosphorylated AKT and N-cadherin expression levels in HepG2/5-FU cells in the presence of 5-FU (Fig. 4A).

The reversal effects of KGM on the resistance of HepG2/5-FU cells to 5-FU were also investigated in vivo. The results demonstrated that KGM inhibited the HepG2/5-FU tumor growth in vivo (Fig. 4B); in the control group, the volume of tumors 13 days after 5-FU-treatment were ~4.69-fold higher compared with those at the start of treatment, whereas the volume of tumors 13 days after KGM and 5-FU treatment were <1.94-fold higher compared with those at the start of treatment (Fig. 4C). In addition, the weight of the tumors was significantly lower in the KGM-treated group compared with that in the control group (Fig. 4D).