Apigenin was obtained from Sigma (St. Louis, MO, USA) and was dissolved in dimethyl sulfoxide to a final concentration of 0.1% in media without causing cytotoxicity. Anti-β-actin and CK2α antibodies were obtained from Calbiochem (La Jolla, CA, USA).

The HeLa human cervical cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carslbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco), 100 units/ml penicillin (Gibco) and 100 μg/ml streptomycin (Gibco). In all experiments, the cells were maintained at 37°C, in a 5% CO₂ and 95% air atmosphere. All the experiments were performed on cultures that were 70% confluent. The present study was approved by the ethics committee of Hunan Normal University (Changsha, China).

Single cell suspensions were suspended at a density of 5,000 cells/ml in serum-free DMEM/F12 supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, 20 ng/ml human recombinant epidermal growth factor, 10 ng/ml human recombinant basic fibroblast growth factor, 2% B27 supplement without vitamin A and 1% N2 supplement (Invitrogen Life Technologies, Carlsbad, CA, USA) and seeded into ultra low attachment 6-well plates (Corning Inc., Corning, NY, USA). Suspension cultures were continued for 6 days until tumorspheres were formed. In order to propagate spheres in vitro, the sphere cells were collected by centrifugation (1,000 g for 5 min), dissociated into single-cell suspensions and cultured to permit the regeneration of spheres. Third-generation spheres were used for all subsequent experiments.

To investigate the percentage of single cells capable of regenerating new spheres, cells were plated at a density of 1,000 cells/ml in a 6-well plate in order to obtain new spheres. The total number of tumor spheres was counted after 6 days of culture. Sphere formation efficiency was calculated using the following formula: (Total number of spheres formed / total number of live cells seeded) × 100.

The third-generation spheres were dissociated, as described above, and 100 cells were plated in 150 μl of growth medium in a 96-well culture plate to obtain a single cell per well. Growth medium (20 μl) was added to each well every 3 days. The number of clonal tumor spheres in each 96-well culture plate was evaluated after 6 days of culture.

SFCs from the HeLa cell line and the parental cells were seeded in 96-well plates precoated with Matrigel (Gibco-BRL) at a density of 5,000 cells per well. Cells were exposed to increasing concentrations (10, 20 and 40 μmol/l) of apigenin. After 48 h, MTT reagent (Sigma) was added to each well according to the manufacturer’s instructions. Absorbance was measured at 570 nm using an automated microplate reader (Bio-RAD 550; Bio-Rad, Hercules, CA, USA).

The pcDNA3.1-CK2α or control pcDNA3.1-LacZ plasmid vectors were transfected into HeLa cells or the SFCs of HeLa cells (0.5 μg/ml in a 24-well plate) using Lipofectamine 2000 transfection reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. The cells were resuspended in complete medium (DMEM supplemented with 10% fetal bovine serum; Gibo-BRL) for 48 h. The cells were harvested and western blotting was performed using mouse monoclonal antibodies against βactin and CK2α.

Western blot analysis was performed, as previously described (18). Cells were lysed by incubating in lysis buffer for 20 min at 4°C. The protein concentration was determined using the Bio-Rad assay system (Bio-Rad, Hercules, CA, USA). Total proteins were fractionated using SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Signals were detected using an ECL Advance western blot analysis system (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA).

Statistical analysis and database management was performed using SPSS (version 15.0) software (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. Multiple group comparisons were made using one-way analysis of variance and pairwise comparison was performed using the least squares difference method. P<0.05 was considered to indicate a statistically significant difference.

HeLa cells were plated in stem cell-conditioned culture medium in 6-well plates at a density of 10,000 cells/well, which enabled the formation of discrete colonies. Under these conditions, cells grew as non-adherent, three-dimensional sphere clusters. The HeLa cella maintained in DMEM medium supplemented with 10% fetal bovine serum (monolayer adhere growth cells) and anchorage-independent spheres formed in the HeLa cell line are shown in Fig. 1A and Fig 1B, respectively. The spheres were passaged after 6 days once they had reached ~50 μm in diameter. The HeLa sphere-derived cells were serially passaged for >12 generations, indicating that HeLa sphere-derived cells were fully capable of self-renewal in vitro.

To determine the percentage of CCSLCs in HeLa third-generation spheres, a limiting dilution assay was used to examine the ability of single cells from third-generation spheres to produce new spheres. After 6 days of culture, 31% of the single cells had generated new spheres (Fig. 1C). By contrast, a lower percentage of single cells derived from the HeLa cells could regenerate spheres when compared with the single cells derived from third-generation spheres (Fig. 1D). These results demonstrated that a considerable percentage of single cells derived from third-generation spheres were self-renewing cells that were able to be expanded and maintained in culture as tumor spheres.

It has been reported that cancer stem cells have the characteristics of extensive proliferation and apigenin has been demonstrated to inhibit the proliferative activity of glioma cancer stem-like cells (9). In the present study, the MTT results demonstrated that apigenin (10, 20 and 40 μmol/l) preferentially inhibited the proliferation of SFCs derived from HeLa cells (Fig. 2A), suggesting that apigenin is able to preferentially suppress the proliferative ability of CCSLCs.

Apigenin (10, 20 and 40 μmol/l) reduced the number of spheroids formed in the SFCs of HeLa cells in a concentration-dependent manner (Fig. 2B). These results suggest that apigenin can suppress the self-renewal capacity of CCSLCs.

Previous studies have reported that CK2α is involved in the activation of the Hh and Notch pathways and is associated with the maintenance of cancer stem cell properties (16,17). The present study aimed to compare the status of CK2α protein expression in parental cells and in SFCs. The results demonstrated that the expression of CK2α was higher in the SFCs than in the parental cells (Fig. 3A). In addition, CK2α expression in SFCs was downregulated by apigenin (Fig. 3B).

Western blot analysis demonstrated that upregulation of CK2α by pcDNA3.1-CK2α transfection resulted in overexpression of the CK2α protein in the parental HeLa cells (Fig. 4A). The results from the tumorsphere formation assay revealed that overexpression of CK2α attenuated apigenin-induced downregulation of CK2α protein expression in the HeLa SFCs (Fig. 4B) and also revealed that overexpression of the CK2α protein increased the self-renewal capacity of HeLa cells (Fig. 4C). In addition, . It also partially reduced the inhibition of self-renewal of SFCs by apigenin (Fig. 4D). These results provided mechanistic evidence that apigenin-inhibited self-renewal was, in part, due to inactivation of CK2α in SFCs derived from HeLa cells.