ABCG2 antibody (cat. no. sc-18841) was purchased from Santa Cruz Biotechnology, Inc. IgG-FITC antibody (cat. no. 115-095-003) was purchased from Jackson ImmunoResearch Laboratories, Inc. Propidium iodide/RNase (cat. no. 550825) and Annexin V-FITC/propidium iodide (cat. no. 556547) were purchased from Becton, Dickinson and Company. ADM (cat. no. H33021980) was purchased from Hanhui Pharmaceuticals Co., Ltd.

The HepG2 cell line (cat. no. CL-0103) was obtained from Procell Life Science & Technology Co., Ltd., and was authenticated by STR profiling and analyzed for mycoplasma contamination. The HepG2 cell line was cultured in MEM containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin and streptomycin. Cells were sustained in an incubator at 37˚C with 5% CO₂. An ADM-resistant hepatoma cell line was established from the HepG2 cells by continuous exposure to increasing concentrations of ADM, from 0.01 to 0.1 µg/ml for 3 months. One of the surviving clones was isolated and designated as HepG2/ADM cells.

pcDNA3.1-ABCG2 plasmid [sequences: CDS of ABCG2 (BC021281) gene (494…2,461)] containing ABCG2 cDNA and empty pcDNA3.1 plasmid were purchased from Wuhan Genesil Biotechnology Co., Ltd. Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used as the transfection reagent, according to the manufacturer's instructions, and stably transfected cell clones were selected using 600 mg/l G418 (without penicillin and streptomycin) subsequent to transfection for 72 h. The transfection reagents used were as follows. i) Reagent 1: 5 µl of Lipofectamine^® 2000 was mixed with 245 µl of MEM culture medium (FBS- and antibiotic-free), and then placed at room temperature for 5 min; ii) reagent 2: 4 µl of pcDNA3.1-ABCG2 or pcDNA3.1 plasmid (containing 2 µg plasmid) was mixed with 246 µl of MEM culture medium (FBS- and antibiotic-free) and then placed at room temperature for 5 min. Reagents 1 and 2 were mixed gently, placed at room temperature for 20 min and then added into the wells. The HepG2 cells transfected with pcDNA3.1-ABCG2 or pcDNA3.1 and selected with 600 mg/l G418 for 10 days were termed HepG2/ABCG2 or HepG2/pcDNA3.1 cells, respectively. ABCG2 mRNA and protein expression levels were monitored to ascertain the efficacy and specificity of the transfection using reverse transcription-quantitative PCR and FCM, respectively.

The ADM anticancer drug sensitivity of the HepG2, HepG2/pcDNA3.1, HepG2/ADM and HepG2/ABCG2 cells was determined using the MTT assay. This method is based on the capacity of viable cells to metabolize a yellow tetrazolium salt, MTT, using mitochondrial succinate dehydrogenase, into purple formazan crystals when dissolved in acidified propan-2-ol; the resulting purple solution is then spectrophotometrically measured at 490 nm (14). The cells were seeded into 96-well culture plates at a density of 1x10⁴ cells/ml. The serial concentrations of ADM (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 and 50 µg/ml) were added in a final volume of 200 µl/well. Normal saline (vehicle; containing 0 µg/ml ADM) was used for the control group. Following drug treatment in an incubator at 37˚C with 5% CO₂ for 24 h, the medium was replaced with an equal volume of complete culture medium containing 0.5 mg/ml MTT and incubated at 37˚C with 5% CO₂ for 4 h. The medium was removed, 180 µl DMSO was added to each well and plates were continuously shaken for 10 min at room temperature. The cytotoxic effects of the drugs were determined using the optical density (OD) values from a microplate reader at an absorption wavelength of 490 nm (OD₄₉₀). The inhibitory rate (IR) was calculated using the following formula in order to calculate the half-maximal inhibitory concentration (IC₅₀) of the cells: IR (%)=(1-OD₄₉₀ treated cells/OD₄₉₀ control cells) x100%. The resistance index was determined as the IC₅₀ of the resistant cells (HepG2/ADM or HepG2/ABCG2 cells)/IC₅₀ of the parental cells (HepG2 cells). Each experiment was performed in triplicate wells.

Total RNA of cells was extracted by RNA isolater (Vazyme Biotech) according to the manufacturer's instructions. Following extraction of total RNA from HepG2, HepG2/pcDNA3.1, HepG2/ADM and HepG2/ABCG2 cells, total RNA was reversely transcribed into cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme Biotech) according to the manufacturer's instructions, and PCR amplification was then performed using the cDNA as a template. The thermocycling conditions were as follows: 95˚C for 5 min (pre-denaturation), followed by 40 cycles of 95˚C for 15 sec, 58˚C for 30 sec and 72˚C for 25 sec, and a final cycle of 95˚C for 15 sec, 58˚C for 1 min and 95˚C for 30 sec. SYBR-Green I was used as the fluorescent dye. Real-time PCR was performed using AceQ qPCR SYBR Green Master Mix kit (Vazyme Biotech) according to the manufacturer's instructions. Human GAPDH was used as the internal reference for standardization. The primers used were as follows: ABCG2 forward, 5'-GGTCAGAGTGTGGTTTCTGTAGCA-3' and reverse, 5'-GTGAGAGATCGATGCCCTGCTTTA-3'; and GAPDH forward, 5'-ACCACAGTCCATGCCATCAC-3' and reverse, 5'-TCCACCACCCTGTTGCTGTA-3'. The relative expression level of ABCG2 mRNA was calculated using the 2^-ΔΔCq method (ΔCq=Cq_ABCG2-Cq_GAPDH) (15). Each experiment was performed three independent replicates.

HepG2/ADM, HepG2/ABCG2, HepG2/pcDNA3.1 and HepG2 cells were collected during the logarithmic growth phase. A total of 1 ml of the single-cell suspension (containing 1x10⁶ cells) was prepared, washed with cold PBS once and suspended in 100 µl of 1X binding buffer. Subsequently, 10 µl Annexin V-FITC was added and the mixture was placed on ice for 15 min in the dark. Next, 380 µl of 1X binding buffer and 10 µl propidium iodide were added and the cell mixture was incubated on ice for 15 min in the dark, washed with cold PBS once and suspended with 1 ml PBS. The apoptosis of the cells was measured using an FC500 flow cytometer (Beckman Coulter, Inc.). EXPO32 ADC software (version 1.2; Beckman Coulter, Inc.) was used to analyze the immunofluorescence data and evaluate the apoptotic rate. Each experiment was performed as three independent replicates.

A total of 1 ml of the single-cell suspension (containing 1x10⁶ cells) was prepared, washed with cold PBS twice and fixed with 70% ethanol at 4˚C for 6 h. Following this, the single-cell suspension was washed with PBS twice. The cells were then suspended in 100 µl PBS and 1 ml propidium iodide was added to the suspension for staining at 4˚C for 30 min. Subsequently, the cells were detected using the FC500 flow cytometer. MultiCycle AV software (version 275; Phoenix Flow Systems, Inc.) was used to analyze the cell cycle. The proliferative index (PI) was calculated according to the following formula: PI=(S+G₂/M)/(G_0/1+S+G₂/M) x100%. Each experiment was performed as three independent replicates.

HepG2/ADM, HepG2/ABCG2, HepG2/pcDNA3.1 and HepG2 cells were collected during the logarithmic growth phase. A total of 1 ml of the single-cell suspension (containing 1x10⁶ cells) was first prepared, then washed with cold PBS once and resuspended in 100 µl PBS. A total of 10 µl of ABCG2 antibody (dilution, 1:100; cat. no. sc-18841; Santa Cruz Biotechnology, Inc.) was added to each sample. The suspension was then incubated for 30 min at room temperature, washed with cold PBS once and resuspended in 100 µl of PBS. IgG-FITC secondary antibodies (dilution, 1:100; cat. no. 115-095-003; Jackson ImmunoResearch Laboratories, Inc.) were added to each sample. The suspension was then incubated for 30 min in the dark, washed with cold PBS once and resuspended in 1 ml PBS. The stained cells were analyzed using the FC500 flow cytometer and the data were analyzed using EXPO32 ADC software, with the mean fluorescence intensity representing the expression of ABCG2 protein. Each experiment was performed three independent replicates.

The cellular accumulation and efflux of ADM were analyzed by flow cytometry. HepG2/ADM, HepG2/ABCG2, HepG2/pcDNA3.1 and HepG2 cells (4x10⁵ cells) were incubated with 0.1 µg/ml ADM at 37˚C for 2 h and washed twice with ice-cold PBS. The cells were resuspended in ADM-free complete culture medium and incubated in an incubator at 37˚C with 5% CO₂ for 1 h. The cells were washed with ice-cold PBS and the ADM (the ADM has an intrinsic fluorescence peak at 595 nm) (16) retained in the cells was detected using the FC500 flow cytometer and the data were analyzed using EXPO32 ADC software, with the mean fluorescence intensity representing the ADM content. Each experiment was performed three independent replicates.

All data are presented as the mean ± the SD (n=3) and were statistically analyzed using one-way ANOVA or two-way ANOVA (MTT assay: Two independent variables-ADM concentration and group) followed by Tukey's or Bonferroni's test (SPSS software version 21; IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

Following anticancer drug (ADM) treatment for 24 h, the cell growth inhibitory rate was detected by an MTT assay. As presented in Fig. 1, compared with the control group (0 µg/ml ADM), the HepG2, HepG2/pcDNA3.1, HepG2/ADM and HepG2/ABCG2 cell survival decreased in a dose-dependent manner after treatment with concentrations of ADM ranging from 0.001 to 50 µg/ml. The cell growth inhibitory rate in HepG2/ADM and HepG2/ABCG2 cells was significantly lower than that in HepG2 and HepG2/pcDNA3.1 cells (P<0.01). The IC₅₀ was calculated through the MTT assay. The IC₅₀ of HepG2/ADM, HepG2/ABCG2, HepG2/pcDNA3.1 and HepG2 cells was 11.17±0.59, 12.75±0.47, 0.74±0.01 and 0.72±0.03 µg/ml, respectively. The resistance index of HepG2/ADM and HepG2/ABCG2 cells to ADM was 15.51 and 17.71, respectively. HepG2/ADM and HepG2/ABCG2 cells were resistant to ADM.

The results of the FCM analysis indicated no significant difference in the apoptotic rate of HepG2/ADM, HepG2/ABCG2, HepG2/pcDNA3.1 cells and their parental HepG2 cells (P>0.05; Fig. 2).

The results of the FCM analysis (Fig. 3) indicated that the G₀/G₁ phase population of HepG2/ADM and HepG2/ABCG2 cells decreased significantly compared with that of their parental HepG2 cells and HepG2/pcDNA3.1 cells (P<0.05). The PI of HepG2/ADM and HepG2/ABCG2 cells increased significantly compared with their parental HepG2 cells and HepG2/pcDNA3.1 cells (P<0.05). Compared with HepG2/ABCG2 cells, HepG2/ADM exhibited no significant difference in G₀/G₁ phase and PI (P>0.05); compared with HepG2 cells, HepG2/pcDNA3.1 had no significant difference in G₀/G₁ phase and PI (P>0.05).

The results indicated that the gene and protein expression levels of ABCG2 in the HepG2/ADM and HepG2/ABCG2 cells were significantly higher than those in HepG2 and HepG2/pcDNA3.1 cells (P<0.01), but there was no significant difference between HepG2/pcDNA3.1 and HepG2 cells (P>0.05). Compared with HepG2/ADM cells, the expression levels of ABCG2 gene and protein in HepG2/ABCG2 cells increased significantly (P<0.05), as indicated in Fig. 4.

An ADM efflux effect assay was used to investigate how the HepG2/ADM and HepG2/ABCG2 cells resisted the anticancer agent using FCM (Fig. 5). The cells were incubated at 37˚C with 0.1 µg/ml ADM for 2 h and then without ADM for 1 h. The level of ADM decreased in HepG2/ADM and HepG2/ABCG2 cells compared with HepG2 and HepG2/pcDNA3.1 cells (P<0.05). There was no significant difference between HepG2/pcDNA3.1 and HepG2 cells (P>0.05). Thus, the results showed that the ADM efflux effect of the HepG2/ADM and HepG2/ABCG2 cells was more pronounced than that of the HepG2 and HepG2/pcDNA3.1 cells.