To construct a Hep3B/5-Fu resistant cell subline, 5-Fu concentration was gradually increased in the culture media. Initially, Hep3B cells were grown in a media comprising 500 μg/L of 5-Fu and the medium was refreshed every 3 days. Upon reaching the logarithmic growth phase, the 5-Fu concentration in the cells was doubled with each medium change; this process was repeated continuously. After six months, the Hep3B/5-Fu cells were propagated in a media comprising 20,000 μg/L of 5-Fu to preserve their drug resistance. One week before the experiments, the cells were transferred to DMEM lacking 5-Fu to prepare for subsequent tests. The cells were passaged every 3 to 4 days at a 1:2 split ratio.

The log-phase Hep3B and Hep3B/5-Fu cells (5 × 10³/well) were trypsinized and grown in 96-well plates. After overnight incubation, the media was refreshed with fresh DMEM, and three types of chemotherapy drugs were added. Each drug was serially diluted to achieve six different drug concentrations, with the maximum concentration of each drug as follows: epirubicin (Thermo Scientific, T14A061, Waltham, MA, USA) (6.0 mg/L), 5-Fu (80.0 mg/L), and oxaliplatin (2.0 mg/L). Each concentration was tested in triplicate wells. Additionally, three untreated control wells and three cell-free wells were established. After 48 h of incubation, the drug-containing media was replaced with fresh media, and the cells were cultured for an additional 24 h. According to the CCK8 kit’s instructions (APExBIO, K1018, Houston, TX, USA), serum-free medium (80 μL) and CCK8 reagent (6 μL) were added per well, and after 1 h, the absorbance at a wavelength of 492 nm was assessed via a microplate reader (MULTISKAN FC, Thermo Scientific, Waltham, MA, USA). A modified method was employed to calculate the 50% inhibition rate (IC₅₀) of various chemotherapeutic drugs in both Hep3B/5-Fu and Hep3B cells, as well as the drug resistance index ((RI) = IC₅₀ (Hep3B/5-Fu)/IC₅₀ (Hep3B)).

The experimental groups were categorized as follows: the blank control group (DMEM); Apatinib groups (apatinib at concentrations of 10, 20, and 40 μmol/L); 5-Fu groups (5-Fu at a concentration of 1/2 IC₅₀); and Apatinib and 5-Fu combination groups (5-Fu (1/2 IC₅₀) + apatinib (10, 20, and 40 μmol/L)). Each group was replicated three times. The cellular morphology was examined via a microscope (Ti-S, Nikon Instruments Inc., Melville, NY, USA), and the OD values were determined using the CCK8 assay. The rate of growth inhibition was assessed with the following formula: Cell growth inhibition rate (%) = (OD_{control group} − OD_{experimental group})/OD_{control group} × 100%. The reverse index was calculated as (inhibition rate_{5-Fu+apatinib})/(inhibition rate_5-Fu) ×100%.

The experimental groups were organized as follows: control group; 5-Fu groups (5-Fu at a concentration of 1/2 IC₅₀); Apatinib groups at concentrations of 10 and 20 μmol/L; and combination groups of apatinib and 5-Fu (5-Fu (1/2 IC₅₀) + apatinib (10 and 20 μmol/L)). Cells were harvested and rinsed with PBS twice. Apoptosis was assessed with the help of an Annexin V-FITC/PI apoptosis detection kit (BioLegend, 640914, San Diego, CA, USA), per the manufacturer’s protocol. Apoptosis levels were elucidated by flow cytometry (BD, FACSLyric, Franklin Lakes, Bergen, NJ, USA). The apoptotic cell percentage was calculated via FlowJo VX10 (Tree Star, Inc.) software. The experiments were conducted in triplicate.

One day before transfection, logarithmic growth phase cells were seeded. Hep3B/5-Fu cells were plated in a 6-well plate at a density of 5 × 10⁵ cells per well, using a DMEM culture medium without antibiotics. The cell confluence was aimed to be between 60% and 70% at the time of transfection. The experimental groups were organized as follows: a blank control group, a negative control group, and an NF-κB p65 siRNA group (Thermo Scientific, s11915, Waltham, MA, USA). The experiments were conducted in triplicate. qRT-PCR and western blot analyses were used to verify the efficacy of the NF-κB siRNA transfection and to assess changes in the expression of MDR-related genes and proteins.

Western blotting was employed to assess changes in the level of MDR and NF-κB pathway-related proteins in Hep3B/5-Fu cells treated with apatinib following NF-κB siRNA transfection. The experiment was structured into three groups, treated with 0, 20, and 40 μmol/L of apatinib. Total proteins were extracted after 48 h. The alterations in the expression of MDR and NF-κB pathway-linked proteins were then analyzed. This set of experiments was conducted in triplicate.

Forty-eight BALB/c nude mice, evenly split between females and males, aged 4–6 weeks and weighing 16–23 g, were housed under specific pathogen-free (SPF) conditions. All procedures of mouse experiments were approved (No. [2018] S323) by the Animal Care Committee at Huazhong University of Science and Technology. All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology. The multidrug-resistant liver cancer cell line Hep3B/5-Fu (3 × 10⁶) was added in 200 μL of PBS for subcutaneous inoculation into the mice. Following tumor formation, the maximum (A) and minimum (B) diameters were elucidated with a Vernier caliper. For tumor volume assessment, the following formula was employed: V(mm³) = AB²/2 [18]. After one week, once 100 mm³ tumor size was achieved, the mice were randomly categorized into 8 groups of six each: Control, Apatinib monotherapy (50 mg/kg/day) (19), 5-Fu monotherapy (20 mg/kg, 2 times/week), Oxaliplatin monotherapy (6 mg/kg, 2 times/week), 5-Fu + Oxaliplatin, Apatinib + 5-Fu, Apatinib + Oxaliplatin and Apatinib + 5-Fu + Oxaliplatin.

Mice were euthanized 24 h after the last drug administration. Tumors were excised and measured; some were flash-frozen in liquid nitrogen for Western blot and qRT-PCR analyses, while others were fixed in 100 g/L formaldehyde for immunohistochemical staining.

The levels of MDR-linked genes were assessed in the xenografted tumor tissues of the control, apatinib, 5-Fu, and apatinib + 5-Fu groups. qRT-PCR: Total RNA was extracted from xenografted tumor tissues using the Trizol reagent (15596018, Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol. First-strand cDNA was synthesized from 1 μg of total RNA using the PrimeScript RT reagent Kit (RR036A, Takara, Kusatsu, Shiga, Japan) in a 20 μL reaction volume. Amplification and quantification were performed using the SYBR Green PCR Master Mix (Applied Biosystems, 4334973, Waltham, MA, USA) on a StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Each reaction mixture (20 μL total volume) contained 10 μL of SYBR Green PCR Master Mix, 0.5 μL of each primer (10 μM), 2 μL of cDNA, and 7 μL of nuclease-free water. The thermal cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Melting curve analysis was performed to verify the specificity of the PCR products. The relative expression levels of genes were calculated using the 2^−ΔΔCT method, with GAPDH as the reference gene [19]. The specific PCR primers used are listed in Table 1.

Western blot: Proteins were extracted from homogenized tumor tissues using RIPA buffer (R0278, Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined by the Bicinchoninic Acid (BCA) Protein Assay Kit (1-1KT, Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of protein (30 μg) were separated on 10% SDS-polyacrylamide gels and then transferred onto Polyvinylidene Fluoride (PVDF) membranes (03010040001, Sigma-Aldrich, St. Louis, MO, USA). Membranes were blocked with 5% non-fat milk in TBST (Thermo Scientific, 28360, Waltham, MA, USA) for 1 h at room temperature, followed by incubation with primary antibodies against MDR1, MRP2, LRP, GST-pi, TopoIIα, NF-κB p65, and GAPDH (1:1000 dilution) overnight at 4°C. After washing, the membranes were incubated with HRP-conjugated secondary antibodies (1:2000 dilution) for 1 h at room temperature. GAPDH served as the loading control. Protein bands were visualized using the enhanced chemiluminescence (ECL) system (GE Healthcare, RPN2132, Chicago, IL, USA).

These experiments were performed in triplicate to ensure reproducibility and accuracy.

The excised tumor tissues were fixed in 10% neutral buffered formalin for 24 to 48 h to preserve the tissues adequately. After fixation, tissues were dehydrated in an ascending series of ethanol, cleared in xylene, and then embedded in paraffin wax. The paraffin-embedded tissues were sectioned at 4 micrometers using a microtome (Shenyang Hengsong Technology Co., Ltd., HS-S7220-B, Shenyang, Liaoning, China). Sections were stained using Hematoxylin and Eosin (HE). Hematoxylin was used at a concentration of 1% for nuclear staining, and Eosin was used at a concentration of 0.5% for cytoplasmic staining. Streptavidin peroxidase (SP) method was used for immunohistochemical staining. Tissue sections were incubated with primary antibodies (1:200 dilution, MRP2 (GeneTex, GTX130181, Irvine, CA, USA), P-gp (KA&M BIO, ANT(B)0337, Shanghai, China), GST-pi (Novatein Biosciences, Woburn, MA, USA), LRP (ProSci, PSI-15-516, Poway, CA, USA), Topo IIα (KA&M BIO, ANT(B)0092, Shanghai, China)) followed by an HRP-conjugated streptavidin-biotin complex (used at a dilution recommended by the kit, Thermo Scientific, E40970, Waltham, MA, USA). The expression levels of MRP2, P-gp, GST-pi, LRP, and Topo IIα were evaluated using a semiquantitative scoring system as outlined in reference [20]. This approach ensures a structured assessment of protein expression across the samples.

Data are depicted as the mean ± standard deviation (SD). SPSS 22.0 software (SPSS Inc., Chicago, IL, USA) was employed for statistical analyses. Inter-group comparisons were conducted via the independent sample t-test. For multiple group comparisons, analysis of variance (ANOVA) followed by the least significant difference (LSD) test was utilized. p-value of <0.05 was set as the statistical significance threshold. N = 3 for repeated times for each assay.

To evaluate the impact of apatinib on MDR in liver cancer, we first established an MDR liver cancer cell line. After six months of culture, Hep3B cells were successfully maintained in a medium containing 20,000 μg/L 5-Fu. The morphological changes in the Hep3B/5-Fu cells were observed under an optical microscope (Fig. 1A). Compared to the parental Hep3B cells, the drug-resistant Hep3B/5-Fu cells exhibited reduced cell adhesion and a more elongated, fusiform shape with the formation of pseudopodia. Western blot analysis (Fig. 1B) revealed significant increases in the levels of MDR-related proteins P-gp, LRP, MRP2, GST-pi, and Topo IIα (p < 0.05) in the Hep3B/5-Fu cells compared to Hep3B. This suggests that prolonged exposure to chemotherapeutic agents leads to the overexpression of MDR-related proteins and the development of drug resistance. Furthermore, p-p65 and p-IKB expression was markedly up-regulated in the Hep3B/5-Fu cells compared to Hep3B cells (p < 0.05) (Fig. 1B), indicating an enhanced activation of the NF-κB signaling pathway in the multidrug-resistant cell line.

To further confirm the MDR characteristics of Hep3B/5-Fu cells, a drug sensitivity test was performed. As detailed in Table 2, Hep3B/5-Fu cells demonstrated varying degrees of resistance to different chemotherapeutic agents compared to the parent Hep3B cells. Notably, Hep3B/5-Fu cells exhibited substantial resistance to 5-Fu, with a drug resistance index of 46.14 ± 10.26. The resistance indexes for epirubicin and oxaliplatin were 4.31 ± 0.90 and 6.61 ± 0.78, respectively. These findings confirm the successful establishment of the MDR liver cancer cell line Hep3B/5-Fu.

The inhibition rate of Hep3B/5-Fu cells in 40 μmol/L apatinib alone was (31.39 ± 8.31)%, which was significantly higher than that of the 5-Fu group ((11.65 ± 2.67)%, p < 0.05). When apatinib was combined with 5-Fu, there was a notable increase in efficacy even at a lower concentration of 10 μmol/L, with an inhibition rate of 24.59 ± 0.42%. This rate further increased with higher concentrations of apatinib, reaching (43.68 ± 2.73)% at 40 μmol/L apatinib combined with 5-Fu. The reversal index ranged from 2.19 to 3.86 and increased with the apatinib concentration. The inhibition rate of apatinib combined with 5-Fu was significantly higher than that of 5-Fu (p < 0.05) (Table 3). These findings demonstrate that apatinib can effectively reverse the drug resistance in Hep3B/5-Fu cells.

Apatinib at a concentration of 10 μmol/L enhanced apoptosis in Hep3B/5-Fu cells, with the apoptotic rate increasing as the concentration of apatinib increased. The apoptotic rate of the combination of apatinib and 5-Fu was significantly higher than that of the Apatinib or 5-Fu monotherapy (p < 0.05). Such as the combination of apatinib (20 μmol/L) and 5-Fu resulted in an apoptotic rate of 52.12 ± 3.23%, which was significantly higher than the rates observed in the apatinib alone (20 μmol/L) group at 28.89 ± 1.98% (p < 0.001), and the 5-Fu group at 19.10 ± 1.29% (p < 0.0001) (Fig. 2). Statistical t-test validated that the differences were significant (p < 0.01). This indicates a synergistic effect of apatinib and 5-Fu in promoting Hep3B/5-Fu cell apoptosis.

The results of real-time PCR are depicted in Fig. 3A. In comparison with the blank control and negative control groups, the NF-κB siRNA group showed markedly reduced expression levels of p65. Therefore, successful transfection of NF-κB siRNA was verified. Among the assessed MDR-related genes, MDR1 and LRP showed downregulated mRNA expression in the NF-κB siRNA group (p < 0.01). The difference in MRP2, GST-pi, and Topo IIα expression among the three groups was not statistically significant (p ≥ 0.05). Western blot analysis, depicted in Fig. 3B, also demonstrated that NF-κB siRNA transfection in Hep3B/5-Fu cells led to the downregulation of P-gp and LRP at the protein level, further validating the PCR results.

Western blotting in Fig. 3C revealed substantial reductions in the expression of the MDR-related proteins LRP, MDR1, and GST-pi in the apatinib-treated cohort than the control cohort (p < 0.01). Among these, P-gp exhibited the most pronounced decrease, especially at higher concentrations of apatinib (40 μmol/L). Additionally, there was a noticeable decrease in phosphorylated IκBα (p-IκBα) and p65 (p-p65) levels in the apatinib-treated cohort than the control cohort (p < 0.05). However, the expression differences for MRP2, Topo IIα, total p65, and IκBα between the control and apatinib-treated groups were not statistically significant (p > 0.05). The ratios of phosphorylated to total p65 (p-p65/p65) and IκBα (p-IκBα/IκBα) were significantly lower in the apatinib-treated groups (p < 0.01), indicating a significant inhibition of NF-κB pathway activation by apatinib.

Prior to drug administration, the tumor volumes were recorded for the control group and the seven treatment groups, with values of 155.43 ± 46.79 mm³, 152.49 ± 66.80 mm³, 149.72 ± 27.78 mm³, 168.50 ± 32.89 mm³, 162.37 ± 44.54 mm³, 173.49 ± 38.05 mm³, 151.24 ± 32.43 mm³, and 179.94 ± 35.92 mm³, respectively. No statistically significant differences were observed among these initial measurements (p ≥ 0.05). However, as the drug administration period progressed, divergences in tumor volumes among the eight groups became more pronounced, with measurements taken 24 h after the final drug administration.

During the treatment period, tumor volumes were assessed and documented every three days. The resulting tumor growth curves, as illustrated in Fig. 4A, demonstrated that the groups receiving combined treatment (apatinib plus chemotherapy) exhibited significantly lower tumor growth rates compared to those receiving chemotherapy alone (p < 0.05). Notably, the group treated with the triple-drug combination of Apatinib, 5-Fu, and Oxaliplatin showed the slowest growth rate and the smallest tumor volumes.

The nude mice were euthanized at the end of the treatment schedule, and the tumors were harvested for measurement. The tumor volume in the Apatinib + 5-Fu + Oxaliplatin group was substantially smaller than in all other groups, corroborating the trends observed in the tumor growth curves (Fig. 4B). This suggests a superior efficacy of this combination therapy in reducing tumor growth.

Real-time PCR results depicted in Fig. 5A show that, compared with the control and apatinib-treated groups, the 5-Fu group exhibited markedly increased MDR-related gene expression, including MDR1, LRP, MRP2, and GST-pi (p < 0.05). In contrast, the combination treatment group (apatinib + 5-Fu) demonstrated significantly reduced expression levels of these genes when compared to the 5-Fu group alone. The expression of Topo IIα did not show significant differences among the four groups (p > 0.05). To validate these changes at the protein level, Western blot analysis was conducted. The results, shown in Fig. 5B, are consistent with the real-time PCR data, indicating that apatinib effectively suppressed the expression of P-gp, LRP, MRP2, and GST-pi proteins (p < 0.05).

Further confirmation came from immunohistochemistry analysis, with results presented in Fig. 5C. These results demonstrate that the combined treatment of apatinib and 5-Fu significantly downregulated the expression of P-gp, MRP2, LRP, and GST-pi compared to 5-Fu alone, corroborating the findings from both the Western blot and real-time PCR analyses. This comprehensive approach confirms the modulation of drug resistance genes by apatinib in combination with 5-Fu, highlighting its potential to enhance the efficacy of chemotherapy by overcoming drug resistance.