Potentilla chinensis was collected during July–August 2014 from a local region of Henan, China. The plant material was confirmed by a well-known taxonomist. The roots of Potentilla chinensis were thoroughly washed with tap water, shade dried and then chopped into small pieces. Ethanol (95%) was used for hot extraction, which was conducted for 3 h using a soxhlet extraction apparatus. The extract was then concentrated under reduced pressure in a rotary evaporator at 45°C and was then kept in a refrigerator at 4°C prior to use.

RPMI-1640 growth medium (Hangzhou Sijiqing Biological Products Co., Ltd., Hangzhou, China), minimum essential medium (MEM), fetal calf serum (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), trypsin, penicillin, MTT, streptomycin, dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS) were used in this study. The MTT kit was obtained from Roche Diagnostics (Indianapolis, IN, USA). Annexin V-Fluorescein Isothiocyanate (FITC)-Propidium Iodide (PI) Apoptosis Detection kit was purchased from Sigma-Aldrich (St. Louis, MO, USA). Hoechst dye was purchased from Sigma-Aldrich. All other chemicals and solvents used were of the highest purity grade. Cell culture plastic ware was purchased from BD Biosciences (San Jose, CA, USA).

The MG63 human osteosarcoma cell line and fR-2 normal epithelial cell line were obtained from Shanghai Institute of Cell Resource Center of Life Science (Shanghai, China). All cells were grown in a humidified 5% CO₂ atmosphere at 37°C in an incubator, and cultured in RPMI-1640 medium supplemented with 10% heat-inactivated newborn calf serum, 100 IU/ml penicillin and 100 µg/ml streptomycin.

Inhibition of cell proliferation of the extract was measured using an MTT assay. Briefly, MG63 and fR-2 cells were plated in 96-well culture plates (1×10⁵ cells/well) separately. After 24 h incubation, cells were treated with the ethyl acetate extract of Potentilla chinensis (EEPC) (0, 5, 10, 20, 40, 80 and 150 µg/ml, eight wells per concentration) for 24 or 48 h. MTT solution (10 mg/ml) was then added to each well. After 4 h incubation, the formazan precipitate was dissolved in dimethyl sulfoxide (100 µl) and then the absorbance was measured in an enzyme-linked immunosorbent assay reader (Thermo Molecular Devices Co., Union City, CA, USA) at 570 nm. The cell viability ratio was calculated by the following formula: Inhibitory ratio (%) = (OD control−OD treated) / (OD control) × 100.

Phase contrast microscopy was conducted to assess the morphological alterations in the MG63 osteosarcoma cancer cells. The cells were incubated for 24 h and treated with EEPC extract at various concentrations (0, 40, 80 and 150 µg/ml). Control cells treated with 0.5% DMSO alone were also included. The morphological changes, characteristic of apoptosis or necrosis, were observed and the images were captured under an inverted light microscope (Olympus America, Inc., Center Valley, PA, USA) after 24 and 48 h.

To establish and confirm cells undergoing apoptosis, an Annexin V binding assay was performed through flow cytom-etry. Briefly, MG63 cancer cells were treated with the EEPC extract (0, 40, 80 and 150 µg/ml) for 48 h, and then treated and untreated cells were harvested by trypsinization. Harvested cells were then incubated in Annexin V-FITC (50 ng/ml) and PI (50 µg/ml), at room temperature for 20 min in the dark, and analyzed using a FACS Calibur flow cytometer (BD Biosciences) taking a minimum of 20,000 cells in each sample.

MG63 cells (5×10⁶) were seeded in 60-mm dishes and subjected to various concentrations (0, 40, 80 and 150 µg/ml) of the EEPC for 48 h. Floating and adherent cells were collected by trypsinization and washed three times with PBS. Cells were incubated in 70% ethanol at −20°C overnight, treated with 20 µg/ml RNase A and stained with 2.0 µg/ml PI. Finally the stained cells were analyzed by Flow cytometry at a wavelength of 488 nm using a FACS Calibur instrument (BD Biosciences) equipped with CellQuest software, version 3.3 (BD Biosciences).

The MG63 cells (1×10⁵ cells/dish) were plated in a 6-cm dish and then subjected to treatment with various concentrations (0, 40, 80 and 150 µg/ml) of the EEPC for 48 h. After drug treatment, the cells were washed with ice-cold PBS and resuspended in lysis buffer (25 mM Tris-HCl, pH 7.4, 5 mM EDTA and 0.6% SDS) with 1.0 mg/ml RNase A for 15 min at 50°C. Proteinase K was added and the cells were incubated overnight. Separation of DNA was conducted using 2% agarose gel and detected under UV light after staining with ethidium bromide.

This assay was performed using a standard method (10). Cells (1×10⁵ cells/ml) were seeded in a 6-well plate and incubated at 37°C until a monolayer of 95–100% confluent cells was obtained. Subsequent to 12 h of starvation, a 500 ml pipette tip was used to create a straight cell-free wound. Each well was washed twice with PBS to remove any debris and then exposed to various concentrations of EEPC extract (0, 40, 80 and 150 µg/ml) in a medium. After 48 h of incubation, the cells were fixed and stained with 5% ethanol containing 0.5% crystal violet powder for 20 min, and randomly selected fields were photographed under a light microscope. The number of cells that migrated into the scratched area were counted.

All data were derived from at least three independent experiments. The results are expressed as the mean ± standard deviation. Differences between groups were analyzed using Student's t-test. GraphPad Prism, version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analyses. P<0.05 was considered to indicate a statistically significant difference.

The EEPC was evaluated for antiproliferative activity using the MTT assay against the MG63 human osteosarcoma cancer cells and an fR-2 normal cell line (epithelial cell line) for 24 and 48 h (Figs. 1 and 2). The extract exhibited potent, dose-dependent and selective cytotoxic activity against MG63 cancer cells. With the aim of investigating the toxic effects of the extract on normal cells, the cytotoxic effects of the extract against fR-2 normal epithelial cells was also assessed (Fig. 2). The results showed that the extract resulted in decreased cyto-toxicity towards the normal cell line, and as such was specific towards the cancer cells. The effect of the EEPC extract on the MG63 osteosarcoma cancer cell growth was estimated by MTT assay conducted for two different time durations, 24 and 48 h. The results showed that the cytotoxic effect of the extract showed dose and time-dependence.

Morphological analysis using inverted light microscopy revealed that EEPC extract induced growth inhibition and apoptosis in MG-63 osteosarcoma cancer cells. As shown in Fig. 3A–D, the number of cells in the control and the ones treated with different concentrations of EEPC extract increased. The cells with higher doses revealed that cellular shrinkage and blebbing occurred. This effect was shown to be associated with EEPC extract dose. The number of cells with altered morphology increased with EEPC extract concentration.

Translocation of phosphatidylserine to the exterior surface of the plasma membrane is a unique feature of early apoptosis, which can be recognized and detected by binding of Annexin V-FITC. If cell death occurs, fragmented and damaged DNA becomes permeable for binding with PI. After cells are stained with Annexin V in tandem with PI, this reagent enters the cell only when the plasma cell membrane is damaged. In this study, flow cytometry revealed that in the extract-treated cells, a higher number of cells were positive for Annexin V compared with control (no drug treatment) (Fig. 4). The percentage of viable cells was low at lower concentration of the extract. However, at higher dosages, the total number of apoptotic cells considerably increased following treatment with 80 and 150 µg/ml doses of the extract. When the cells were treated with 40, 80 and 150 µg/ml of the EEPC extract for 48 h, the average proportion of Annexin V-staining positive cells (total apoptotic cells) markedly increased from 5.6% in control to 24.2, 38.8 and 55.7%, respectively. Thus, this assay allows a quantitative estimation of the apoptotic cell death following drug exposure.

Apoptosis and the cell cycle are closely related biochemical processes, and any disruption in cell cycle progression may finally lead to apoptotic cell death. In order to have a mechanistic indication of the growth inhibitory effect exerted by the extract in MG-63 cancer cells, flow cytometry analysis was conducted to identify whether the extract induces cell cycle arrest in this cell line. The results revealed that EEPC extract induces sub-G1 cell cycle arrest and increases the fraction of MG-63 apoptotic cells. To determine the distribution of EEPC extract-treated MG-63 cells in different phases of the cell cycle, DNA content in cells was detected by PI staining and flow cytometry. The extract induced sub-G1 cell cycle arrest with a substantial increase in the number of apoptotic cells. The results showed that treatment with different concentrations of the extract for 48 h led to an increase in the population of cells in the sub-G0/G1 phase (apoptotic population) (P<0.01) (Fig. 5A–D). This upsurge in the sub-G1 population was accompanied by a corresponding reduction of the cells in the s-phase and G2/M phases of the cell cycle. As compared with the control (Fig. 5A), extract treated (Fig. 5B–D) cells showed a significant proportion of apoptotic cells.

Besides the morphological changes to extract-treated MG-63 cells, DNA fragmentation was also examined by observation of the formation of DNA ladder. As shown in Fig. 6, the DNA ladder appeared to be more evident with the increasing extract concentration; however, no DNA fragments were observed in the control group (Fig. 6, 0 µg/ml). However, 40, 80 and 150 µg/ml doses of the extract after 48 h exposure led to a substantial increase in DNA fragmentation (Fig. 6, right panel). The DNA fragmentation is a hallmark of apoptosis, further confirming that the EEPC extract induced cell death through apoptosis.

In this experiment, the effect of EEPC extract on the migration in MG-63 osteosarcoma cells was examined. Confluent cells were scratched and then treated with EEPC extract in complete medium for 48 h. The number of cells that migrated into the scratched area was captured (magnification, ×40; inverted light microscope; Olympus America, Inc., Center Valley, PA, USA) and calculated as a percentage (%) of migration. As shown in Figs. 7 and 8, EEPC extract significantly reduced MG-63 cell migration in a concentration-dependent manner.