The human cervical cancer cell lines, C-33A (cat. no. HTB-31), HT-3 (cat. no. HTB-32), Me180 (cat. no. HTB-33) and MS751 (cat. no. HTB-34), were obtained from American Type Culture Collection (Manassas, VA, USA). HeLa cells, derived from human cervical carcinoma, were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin and penicillin, followed by incubation at 37°C in a 5% CO₂ incubator. The C33A cells were cultured in DMEM containing l-glutamine, 10% FBS, 1% non-essential amino acids and 1% sodium pyruvate. The HT-3 and Me180 cells were cultured in McCoy’s medium (Sigma-Aldrich, St. Louis, MO, USA) with l-glutamine and 10% FBS. The MS751 cells were cultured in Eagle’s minimum essential medium (Sigma-Aldrich) with l-glutamine, 10% FBS, 1% non-essential amino acids and 1% sodium pyruvate.

Ginsenoside-Rg5 (purity>95%) was provided by Dr WM Zhao at the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences (Shanghai, China). Dimethylsulfoxide, Trypan blue, low melting agarose, 4′,6-diamidino-2-phenylindole (DAPI), normal melting agarose, 2-amino-2-(hydroxymethyl)-1,3-propanediol, sodium dodecyl sulfate, ethylenediaminetetraacetic acid, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-methyl-N-nitro-N-nitrosoguanidine (MNNG, inducing DNA damage), Tween 20 and paraformaldehyde were obtained from Sigma-Aldrich. Propidium iodide (PI) (21) and DMEM were obtained from Gibco Life Technologies (Carlsbad, CA, USA). The Apoptosis Detection kit I was obtained from BD Pharmingen San Diego, CA, USA). Triton X-100, ethidium bromide, FBS, xylene cyanol and bromphenol blue were obtained from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China).

The cells were seeded into 96-well plates at a density of 5×10⁴ cells per well and left overnight for cell adherence. Cell viability was determined between 12 and 48 h in the presence or absence of metformin using an MTT assay (22). Plates were read using an Automated Microplate Reader (Multiskan EX; Lab Systems, Helskinki, Finland) at a test wavelength of 570 nm.

The HeLa and MS751 cells were treated using ginsenoside-Rg5 at concentrations of 0, 2.5 or 5 μM for 12 or 24 h, respectively. Cells treated with 10 μM MNNG for 24 h were used as a positive control (CT). The treated cells were then harvested and washed with phosphate-buffered saline (PBS). DNA was extracted using a Wizard Genomic DNA Purification kit (Promega Corporation, Madison, WI, USA) (23). DNA fragmentation was detected by electrophoresis on a denaturing urea polyacrylamide gel, which was stained using silver nitrate solution (24).

Cell apoptosis was determined using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit I (BD Pharmingen). Briefly, the cells were treated with ginsenoside-Rg5 and MNNG at concentrations between 0 and 5 μM and at 10 μM, respectively, for 24 h. The cells were then washed twice with cold PBS and resuspended in 500 μl binding buffer at a concentration of 5×10⁵ cells/ml. Subsequently, 5 μl annexin V-FITC solution and 5 μl PI (1 mg/ml) were added to the cells at 37°C for 30 min. The cells were analyzed using flow cytometry within 1 h. The number of apoptotic cells were counted and represented as a percentage of the total cell count.

The HeLa and MS751 cells were treated with 0, 0.625, 1.25, 2.5 or 5 μM ginsenoside-Rg5 for 24 h and were then collected, washed and suspended in PBS (pH 7.4). Subsequently, 30 μl cell samples (1×10⁴ cells) were suspended in 110 μl of 1% molten low-melting-point agarose at 37°C (26). The monosuspension was cast on a microscopic slide that had been covered with a layer of 0.8% regular-melting-point agarose. Images were captured using an Olympus BX53 fluorescent microscope (Olympus Corporation, Tokyo, Japan) using a filter of 515–560 nm. The extent of DNA migration was determined using an image analysis system (CASPLab; www.casp.of.pl) and the tail length, indicating DNA migration from the nucleus, and tail moment (migrated DNA in the tail multiplied by the tail length) were recorded. All cells treated with MNNG (10 μM) were included as positive controls.

The phosphorylation of histone H2AX as a marker of DNA double-strand breaks (DSBs) was performed, as previously described (24). The HeLa and MS751 cells (5×10⁵) were seeded into six-well culture plates and treated with 0, 0.625, 1.25, 2.5 or 5 μM ginsenoside-Rg5 and 10 μM MNNG for 24 h. Following treatment, the cells were fixed in 4% paraformaldehyde for 15 min, washed with PBS (pH 7.4) and 0.1% Tween 20 and permeabilized in 1% Triton-X 100 for 30 min. Following inhibition with fetal bovine serum (Gibco Life Technologies, Carlsbad, CA, USA) for 60 min, the cells were incubated with rabbit monoclonal anti-gH2AX antibodies (1:1,500; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C and conjugated with Alexa594-conjugated anti-rabbit secondary antibodies (1:360, Cell Signaling Technology, Inc.) for 60 min. For nuclear staining, 1 mg/ml DAPI was added to the cells and the cells were incubated for 15 min. The cells were then mounted in antifade media and images were captured using an Olympus BX53 fluorescent microscope (Olympus Corporation). The objectives were set at wavelengths of 594 nm for γH2AX and 350 nm for DAPI.

All values are expressed as the mean ± standard error of the mean. The data were analyzed using one-way analysis of variance followed by Student-Newman-Keuls test for multiple comparisons. The Newman-Keuls multiple comparisons test was applied, and P<0.05 and P<0.01 were considered to indicate statistically significant differences.

To determine whether ginsenoside-Rg5 exerted cytotoxic effects on the cervical cancer cells, the cervical cancer cell lines were exposed to various concentrations of ginsenoside-Rg5 and the cell viability was assessed. Although the sensitivities to the treatment varied depending on the cell line, ginsenoside-Rg5 treatment at concentrations ranging from 0.625 to 20 μM for 48 h resulted in concentration-dependent cytotoxicity in the cervical cancer cells (Fig. 2A). The half-maximal inhibitory concentration (IC₅₀), the concentration at which 50% of the cells survive, values of the HeLa and MS751 cells were between 2.5 and 10 μM, while the IC₅₀ values of the C-33A, HT-3 and Me180 cells were all higher than 20 μM (Fig. 2A). To further validate the effects of ginsenoside-Rg5, the cells were exposed to 5 μM ginsenoside-Rg5 for 12, 24 or 48 h. Ginsenoside-Rg5 reduced the viability of the cervical cancer cells in a time-dependent manner (Fig. 2B). The HeLa and MS751 cells had a higher sensitivity to the treatment compared with the C-33A, HT-3 and Me180 cells. Thus, these results suggested that ginsenoside-Rg5 possessed a cytotoxic function in certain cervical cancer cell lines, including HeLa and MS751.

The HeLa and MS751 cells were significantly more sensitive to ginsenoside-Rg5. Therefore, to determine whether the loss in cell viability induced by ginsenoside-Rg5 was associated with apoptosis, ginsenoside-Rg5-induced apoptosis was examined in these sensitive cell lines using DNA fragmentation analysis and flow cytometry. As shown in Fig. 3A, ginsenoside-Rg5 induced a ladder-like pattern on the urea polyacrylamide gel electrophoresis (PAGE). The DNA in the untreated cells remained intact. At concentrations of 2.5 and 5 μM, ginsenoside-Rg5 led to concentration- and time-dependent increases in DNA fragmentation. In addition, in HeLa and MS751 cells, the fraction of apoptotic cells increased markedly when the cells were treated with 1.25–5 μM ginsenoside-Rg5 (Fig. 3B). Taken together, these results suggested that the anticancer activity of ginsenoside-Rg5 was mediated, in part, by the induction of apoptosis.

DNA strand breaks were induced by ginsenoside-Rg5, as demonstrated by the comet assays. The alkaline comet assay revealed that DNA fragments migrated to form comet-like images indicative of DNA damage (27). In the control cells, high-density DNA was observed in the comet heads with smooth margins and intact nuclei. The comet frequencies in the HeLa and MS751 cells were 6.1 and 4.5%, respectively. In the cells treated with ginsenoside-Rg5, the DNA comets exhibited broom-shaped tails. The percentages of comet-positive HeLa and MS751 cells significantly increased at concentrations between 0 and 5 μM compared with the negative control (Fig. 4).

The mean ± SEM of the tail length and tail moment in the HeLa and MS751 cells are shown in Table I. These results indicated that the cells, which were exposed to different concentrations of ginsenoside-Rg5, exhibited significantly higher DNA damage (P<0.01) compared with the control samples. In these cell lines, ginsenoside-Rg5 significantly increased the tail length (P<0.01) and tail moment (P<0.01) when used at concentrations of 1.25 to 5 μM.

The phosphorylation of histone H2AX foci formation has been suggested as a sensitive way to detect DNA DSBs (27). A threshold of four or more γH2AX foci/cell is optimal for determining the extent of DNA damage (28). Immunofluorescent images of histone H2AX phosphorylation in the γH2AX-stained HeLa and MS751 cells are shown in Fig. 5A. Ginsenoside-Rg5 caused a concentration-dependent induction of γH2AX foci. In the control, the HeLa and MS751 cells had few γH2AX-positive foci in the nuclei and there were ~5.5% cells containing over four foci (Fig. 5B). All treatments with ginsenoside-Rg5 and MNNG induced the formation of foci and increased the percentages of γH2AX-positive cells. In addition, ginsenoside-Rg5 and MNNG exhibited distinct concentration-dependent effects (P<0.01) on the formation of γH2AX foci in the HeLa and MS751 cells (Fig. 5B).