The human HCC SMMC-7721 cell line was obtained from the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen Life Technologies, Carlsbad, CA, USA) in an incubator containing 5% CO₂ at 37°C. Casticin was purchased from Chengdu Biopurify Phytochemicals Ltd. (purity, ≥98%; Chengdu, China) and dissolved in dimethyl sulfoxide (DMSO) as a 10 mmol/l stock solution, and diluted in a medium to the indicated concentration before use. Trypsin and DMSO were purchased from Amersco Inc. (Solon, OH, USA).

Cells were labeled with primary CD133/1 antibody [mouse immunoglobulin 1 (IgG1), 1 μl per million cells; Miltenyi Biotec, Bergisch Gladbach, Germany], subsequently magnetically labeled with rat anti-mouse IgG1 microbeads (20 μl per 10 million cells; Miltenyi Biotec) and separated on MACS LS column (Miltenyi Biotec). All procedures were performed according to the manufacturer’s instructions. The purity of sorted cells was evaluated by flow cytometry (FCM) and western blot analysis. The FCM was performed with Epics Altra Cell Sorter (Beckman Coulter, Inc., Pasadena, CA, USA) using CD133/1 primary antibody (Miltenyi Biotec) and phycoerythrin-conjugated secondary antibody (Sigma-Aldrich, St. Louis, MO, USA). The CD133⁺ cells and the parental cells were collected and washed to remove serum, and then suspended in serum-free DMEM/F12 supplemented with 20 ng/ml human recombinant epidermal growth factor (Invitrogen Life Technologies), 20 ng/ml human recombinant basic fibroblast growth factor (Invitrogen Life Technologies), 2% B27 supplement without vitamin A (Invitrogen Life Technologies), 0.4% bull serum albumin (Invitrogen Life Technologies), 4 ng/ml insulin (Invitrogen Life Technologies), 100 IU/ml penicillin and 100 μg/ml streptomycin. The cells were subsequently cultured in ultra low attachment six-well plates (Corning Inc., Corning, NY, USA) at a density of no more than 2,000 cells/well.

The CD133⁺ sphere-forming cells (SFCs) of the SMMC-7721 cell line were collected by gentle centrifugation, then dissociated with trypsin-EDTA and mechanically disrupted with a pipette. The resulting single cells were centrifuged to remove the enzyme, resuspended in stem cell-conditioned culture medium and allowed to reform spheres. The tumor spheres were passaged every 6 days before they reached a diameter of 50 mm. The volume of HCC spheres was calculated by the following: V = (4/3)πr³, where r is the radius of the sphere. The number and volume of the spheres was observed and compared. To examine the effects of casticin on the sphere formation, the resulting single cells with a density of 2×10³ cells/ml were plated to ultra low attachment six-well plates and supplemented with serum-free medium containing various concentrations of casticin.

Pathogen-free male Balb/c-nu mice (age, 4–5 weeks) were purchased from the Animal Institute of the Chinese Academy of Medical Science (Beijing, China). All animal studies were performed in accordance with the standard protocols approved by the ethics committee of the Hunan Normal University (Changsha, China) and the committee of experimental animal feeding and management. Male Balb/c-nu mice (age, 4–5 weeks) were randomly divided into five groups (four mice per group) and maintained under standard conditions, according to the standard protocols. Cells were suspended in serum free-DMEM/Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) mixture (1:1 volume). Each Balb/c-nu mouse was inoculated subcutaneously with different numbers of CD133⁺ SFCs (5×10², 1×10³, 5×10³, 1×10⁴ and 5×10⁴ cells) in one flank and parental cells (5×10⁴, 1×10⁵, 2×10⁵, 5×10⁵ and 1×10⁶ cells) in the other, respectively. Tumorigenicity experiments were terminated 2 months after cell inoculation. Tumor size was measured with a caliper and the volume was calculated using the following: V (mm³) = L×W²×0.5; where L is the length and W is the width. Harvested tumors were examined and weighed immediately. Moreover, specimens from tumor tissue samples were fixed in 10% neutral-buffered formalin, processed in paraffin blocks and sectioned. The tissue sections were stained with hematoxylin and eosin and examined under light microscopy (IX-70, Olympus Corporation, Tokyo, Japan) for identification of the histopathological changes.

pcDNA3-Twist1 was purchased from GenePharma (Shanghai, China). To generate Twist1-expressing stable transfectants, the parental cells and SFCs of the SMMC-7721 cell line were transfected with pcDNA3-Twist1, and stable clones were selected with 1,000 μg/ml of G418 (EMD Millipore, San Diego, CA, USA) for 4 weeks.

Protein extracts were resolved by 12% SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Bioscience, Shanghai, China) blocked in 5% skimmed milk. Primary antibodies were used as indicated by the manufacturer’s instructions and are as follows: Mouse anti-CD133 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Twist (Cell Signaling Technology, Inc., Beverly, MA, USA), N-cadherin (EMD-Millipore) and E-cadherin (BD Biosciences). After incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Amersham Biosciences, Cardiff, UK), specific protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences). β-actin (Sigma-Aldrich) was used as a loading control.

All values in the figures and text are expressed as the means ± SD. Statistical analyses were performed using SPSS software, version 16.0 (SPSS, Inc., Chicago, IL, USA). Any significant differences among the mean values were evaluated by the Student’s t-test. A two-sided P<0.05 was considered to indicate a statistically significant difference.

In order to isolate LCSCs, SMMC-7721 cells were enzymatically dispersed into single-cell suspensions and incubated using an anti-CD133 antibody (Miltenyi Biotec) and then analyzed by FCM. Following separation on MACS LS column for purity, FCM analysis showed that the CD133-positive cell percentage of CD133⁺ subpopulation was 61.24±2.54%, which was obviously higher than that of CD133⁻ population (1.07±0.34%) and the parental cells (2.72±0.46%), indicating our immunomagnetic separation system was efficient (Fig. 1A).

We performed the tumor sphere formation assay with stem cell-conditioned medium to enrich and expand LCSCs from sorted CD133⁺ cells of the SMMC-7721 cell line. In the case of inoculation of 2,000 cells per well, an increased number of spheres formed in the group of CD133⁺ cells, while at the same time as sphere formation, the sphere volume of CD133⁺ cells was greater than that of the parental cells (Fig. 1B and C).

To examine the tumor initiating capability, the male Balb/c-nu mice were transplanted with various amounts of CD133⁺ SFCs of the SMMC-7721 cell line and parental cells. Our results demonstrated that as few as 1,000 CD133⁺ SFCs were sufficient to induce tumor development, whereas, at least 2×10⁵ SMMC-7721 parental cells were necessary to consistently generate a tumor in the same model and required a longer time period (Table I). Hematoxylin and eosin staining indicated that CD133⁺ SFCs formed similar histological features as the tumor xenografts in the parental cells (Fig. 1D). These results demonstrated that CD133⁺ SFCs sorted from the SMMC-7721 cell line were highly tumorigenic and possessed LCSC properties.

EMT is a key process during the metastasis of LCSCs. Therefore, we sought to examine whether morphological changes existed between LCSCs and parental SMMC-7721 cells cultured adherently in vitro. As shown in Fig. 2A, LCSCs exhibited a spindle-like shape, while parental cells displayed a cobble-stone-like phenotype. However, treatment with 1.0 μmol/l casticin suppressed EMT in LCSCs as morphological changes from a spindle-like shape to a cobble-stone-like appearance were observed.

Moreover, similar results were further confirmed by western blotting using specific antibodies against EMT-relative markers. As shown in Fig. 2B, CD133⁺ SFCs expressed a higher N-cadherin protein level, which is typically associated with mesenchymal cells and a lower expression of epithelium-associated E-cadherin protein. Casticin, at increasing concentrations, induced upregulation of the epithelial marker, E-cadherin, and downregulation of the mesenchymal marker, N-cadherin, after treatment for 24 h in CD133⁺ SFCs derived from the SMMC-7721 cell line (Fig. 2C). These results demonstrated that casticin possessed inhibitory effects of EMT in LCSCs purified from the SMMC-7721 cell line.

The transcription factor, Twist, was identified as the critical molecule of EMT (27,28). Based on our results that casticin inhibited EMT in LCSCs, we further investigated the effects of casticin on the expression of Twist. Twist was highly expressed in CD133⁺ SFCs (Fig. 3A). Western blot analysis indicated that the protein levels were downregulated following treatment with casticin (0.0, 0.1, 0.5 and 1.0 μmol/l) in CD133⁺ SFCs (Fig. 3B).

Casticin inhibited EMT of CD133⁺ SFCs, while the overexpression of Twist prevented casticin-induced inhibition of EMT. We transfected the plasmid, pCDNA3.1-Twist, and the negative control, pCDNA3.1, into CD133⁺ SFCs derived from SMMC-7721 cells. As shown in Fig. 4A, compared with the negative control vector, pCDNA3.1, CD133⁺ SFCs transfected with pCDNA3.1-Twist significantly overexpressed Twist. Furthermore, as shown in Fig. 4B and C, the overexpression of Twist attenuated casticin-induced upregulation of E-cadherin and downregulation of N-cadherin, respectively. This tumor sphere-forming experiment showed that the ectopic expression of Twist in this cell line promoted tumor sphere formation. We also treated CD133⁺ SFCs of SMMC-7721 cells transfected with pCDNA3.1-Twist with 1.0 μmol/l casticin. As shown in Fig. 4D, after the addition of casticin, the sphere-forming ability of CD133⁺ SFCs in this subpopulation was greater than that of the control group. Taken together, the overexpression of Twist partly reduced the inhibitory effect of casticin on EMT of CD133⁺ SFCs. These results further supported that the downregulation of Twist expression may contribute to the inhibitory effects of casticin on EMT of LCSCs derived from the SMMC-7721 cell line.