Normal liver tissues and HCC tissues used in this study were obtained between June 2016 and July 2017 from 48 HCC patients (aged 47.36±4.57; 36 male and 12 female patients) who had been treated with 20 mg/kg docetaxel (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) intravenously for a week at our hospital (Department of Oncology, Jingjiang People's Hospital, Jingjiang China). Informed consent was obtained from all patients. All experimental protocols were approved by the Institutional Ethics Committee of Jingjiang People's Hospital, Jiangsu, China (no. 2018-122).

Human normal liver stem cells and HCC stem cells were isolated from liver tissues of HCC patients at our hospital according to a previous study (26). Briefly, cell suspensions were centrifuged at 300 × g for 10 min and cell pellets were resuspended in 300 µl of buffer/10⁸ total cells after aspirating the supernatant completely. Then, 100 µl of FcR Blocking Reagent/10⁸ total cells and 100 µl of CD133/CD44/CD24 MicroBeads/10⁸ total cells were added, mixed well and incubated for 30 min in the refrigerator. Cells were washed by adding 1–2 ml of buffer/10⁸ cells and centrifuged at 300 × g for 10 min. An appropriate MACS Column and MACS Separator was chosen according to the number of total cells and the number of CD133^+/CD44⁺/CD24⁺ cells. The column was placed in the magnetic field of a suitable MACS Separator, and prepared by rinsing with 500 µl buffer MS. The cell suspension was applied onto the column and washed with the appropriate amount of buffer. Unlabeled cells that passed through and combined with the effluent from the MS step were collected. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 10 mg/ml streptomycin (Sigma-Aldrich; Merck KGaA) in an incubator with a humidified atmosphere of 5% CO₂ at 37°C.

Cell viability was assessed using an MTT assay (Bestbio Biotechnology, Shanghai, China). Briefly, human HCC stem cells at a concentration of 2×10³ cells/well were seeded in 96-well flat-bottomed tissue culture plates (Corning Inc., Corning, NY, USA) for 24 h. Following two washes with phosphate-buffered saline (PBS), cells were incubated in 100 µl culture medium containing 50 nM docetaxel for 12, 24 and 48 h at 37°C prior to the MTT assay. Then, a total of 10 µl MTT and 100 µl culture medium was added to each well, and following incubation for 1 h at 37°C, the optical densities of the samples were measured directly using a spectrophotometric microplate reader (Beyotime Institute of Biotechnology, Haimen, China) at a wavelength of 490 nm.

The apoptotic cells were identified by the terminal-deoxynucleotidyl transferase mediated nick end-labeling (TUNEL) apoptosis assay kit (KeyGen Biotech Co., Ltd., Nanjing, China) according to manufacturer's instructions. Cells at a density of 2×10⁴/ml were cultured in 10% FBS-containing DMEM with 50 nM docetaxel for 24 h and harvested and washed twice with cold PBS by gentle shaking. Resuspended cells were added to 1X binding buffer and the cell density was adjusted to 200,000-500,000/ml. Cell apoptosis was analyzed using a FACScan flow cytometric apparatus (BD Biosciences, San Jose, CA, USA) and the percentage of apoptotic cells was analyzed using FlowJo 7.6.1 software (TreeStar, Inc., Ashland, OR, USA).

The expression of CD133 and SOX2 in human HCC stem cells was assessed by RNA preparation and quantitative reverse transcription-polymerase chain reaction (RT-PCR). Total cellular RNA isolation using TRIzol reagent and cDNA synthesis using Takara PrimeScript II First Strand cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.) was conducted according to the manufacturers' instructions. Specific primer sequences were synthesized in BioSune Biological Technology Corp. (Shanghai, China), and the sequences of the primers were as follows: CD133 forward, 5′-CCATACCTAGGTCCCCGTCC-3′ and reverse, 5′-TTCACTCAAGGCACCATCCC-3′; SOX2 forward, 5′-AACCAGCGCATGGACAGTTA-3′ and reverse, 5′-GACTTGACCACCGAACCCAT-3′; GAPDH forward, 5′-AATGGGCAGCCGTTAGGAAA-3′ and reverse, 5′-GCGCCCAATACGACCAAATC-3′. Data was analyzed using Bio-Rad CFX Manager 1.6 Software (Bio-Rad Laboratories, Inc., Hercules, CA, USA.

Following the treatment with 50 nM docetaxel for 6 h, the cells were incubated for an additional 24 h prior to the collection of cells for protein extraction. The examination of the expression levels of CD133, SOX2, PI3K, AKT and p-AKT was then performed separately. Total protein was extracted using Total Protein Extraction kit (Sigma-Aldrich; Merck KGaA). Total protein was quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology) and 30 µg protein/lane was separated via SDS-PAGE on a 6% gel and run on a 10% gel. The separated proteins were subsequently transferred to nitrocellulose (NC) filter membrane and blocked with Tris-buffered saline (TBS) containing 5% milk powder without fat and 0.05% Tween-20. Antibodies of CD133 (dilution 1:1,000; cat. no. sc-33182), PI3K (dilution 1:1,000; cat. no. 4922), AKT (dilution 1:1,000; cat. no. sc-9272) and p-AKT (dilution 1:1,000; cat. no. sc-33437) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies of SOX2 (dilution 1:3,000; cat. no. 3579) and GAPDH (dilution 1:6,000; cat. no. 5174) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA) and incubated at overnight at 4°C. Secondary antibodies were conserved in our laboratory and incubated at 37°C for 1 h. The protein levels were detected using the chemiluminescence reader, ImageQuant™ LAS4000 (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Relative band ratio was analyzed using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA).

Paraffin-embedded normal liver and cancer liver tissues (3-µm in thickness) were prepared and immunohistochemistry was performed as previously described (27). Primary antibodies against CD133 (dilution 1:500; Santa Cruz Biotechnology, Inc.) and SOX2 (dilution 1:200; Abcam, Cambridge, UK) were used. Data of immunohistochemistry were analyzed using Image-Pro Plus (version 4.1; Media Cybernetics, Rockville, MD, USA).

Human HCC stem cells were treated with PI3K/AKT inhibitor LY294002 at a concentration of 25 µM for 48 h, followed by the addition of 50 nM docetaxel and further incubation for 48 h. Then, cell proliferation, apoptosis, and SOX2 expression were evaluated.

All results were analyzed by one-way analysis of variance (ANOVA) using the SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation (SD). Boferroni's post hoc test was used to determine the statistical differences between the treatment and control groups. P-values were based on the two-sided statistical analysis, and P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1, higher expression of CD133 and SOX2 was detected by immunohistochemistry in HCC samples compared with normal liver tissues, indicating that CD133 and SOX2 were two important factors which may be involved in HCC. Moreover, the expression of CD133 and SOX2 in human HCC tissues was significantly downregulated by docetaxel compared to cells without docetaxel stimulation.

Following 24 and 48 h of cell culture, CD133 expression in normal liver stem cells and HCC stem cells was determined by immunohistochemistry, western blotting and RT-PCR. The results revealed that the percentage of CD133-positive cells was obviously higher in HCC stem cells than that of normal liver stem cells (Fig. 2A). Additionally, compared to normal liver stem cells, CD133 protein expression was highly promoted in HCC stem cells (P<0.01). (Fig. 2B). The assessment of CD133 mRNA expression also supported the conclusion that CD133 expression was increased in HCC stem cells (Fig. 2C).

Human CD133-expressing HCC stem cells were respectively exposed to docetaxel for 12, 24 and 48 h, and the role of docetaxel in human CD133-expressing HCC stem cells was assessed through MTT assay and flow cytometry. The results revealed that compared to the control without docetaxel treatment, docetaxel significantly downregulated cell viability in CD133-expressing HCC stem cells in a time-dependent manner (P<0.01) (Fig. 3A). Conversely, as shown in Fig. 3B, docetaxel significantly promoted apoptosis in CD133-expressing HCC stem cells in a time-dependent manner. Approximately 13.58, 26.13 and 40.24% of cells underwent apoptosis after exposure to docetaxel for 12, 24 and 48 h, respectively, and there was a significant difference between docetaxel-treated groups and the control without docetaxel treatment (P<0.01). The findings above indicated that docetaxel significantly increased the apoptosis in CD133-expressing HCC stem cells.

Since SOX2 expression is associated with HCC, we further detected SOX2 protein and mRNA expression using western blotting and RT-PCR at indicated time-points after docetaxel stimulation. The results demonstrated that SOX2 protein (Fig. 4A) and mRNA (Fig. 4B) expression was significantly inhibited by docetaxel in human HCC stem cells (P<0.05) in comparison with the control.

To further verify the underlying mechanisms of docetaxel in human CD133-expressing HCC stem cells, we explored the expression of PI3K, AKT and p-AKT by western blotting (Fig. 5A). Band analysis indicated that the protein level of PI3K and AKT exhibited no difference between the docetaxel-treated group and non-docetaxel-treated group (P>0.05). Moreover, the p-AKT protein level was comparatively low in docetaxel-treated cells in comparison with the control (P<0.01) (Fig. 5B). It was thus suggested that docetaxel could suppress the PI3K/AKT signaling pathway in human CD133-expressing HCC stem cells.

We next examined the influence of the PI3K/AKT signaling on docetaxel-induced cell apoptosis and -decreased SOX2 expression in human CD133-expressing HCC stem cells. The results revealed that HCC stem cells treated with the PI3K/AKT inhibitor LY294002 exhibited a significant decrease in p-AKT expression compared with untreated cells (Fig. 6A). Cell viability (Fig. 6B) and SOX2 expression (Fig. 6D) were inhibited in both LY294002-treated cells and docetaxel-treated cells. However, there was no difference in cell viability and SOX2 expression between LY294002 plus docetaxel-treated cells and LY294002-treated cells (P>0.05). As demonstrated in Fig. 6C, LY294002 or docetaxel significantly promoted the apoptotic rate in CD133-expressing HCC stem cells (P<0.01). Moreover, the apoptotic rate exhibited no difference between the LY294002-treated group and LY294002 plus docetaxel-treated group (P>0.05). These results indicated that the PI3K/AKT signaling pathway was involved in docetaxel-exerted biological functions in human CD133-expressing HCC stem cells.