Ferruginol (>95% purity by high-performance liquid chromatography) and MTT reagent were purchased from Sigma-Aldrich (Merck KGaA; Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 medium, acridine orange (AO), ethidium bromide (EB) and propidium iodide (PI) were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). Fetal bovine serum (FBS), penicillin and streptomycin were purchased from Tianjin Haoyang Biological Products Co., Ltd. (Tianjin, China). All other chemical reagents used were of analytical grade.

OVCAR-3 human ovary cancer cells were purchased from the Cell Bank of the Basic Medical College of Huazhong University of Science and Technology (Wuhan, China). Cells were cultured in DMEM containing 10% FBS with 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere with 5% CO₂. The cell cytotoxicity induced by ferruginol in these cancer cells was evaluated by an MTT cell viability assay using various doses and incubation durations. Briefly, OVCAR-3 cells were plated at a density of 1×10⁶ cells per well in 96-well plates for 12 h at 37°C. The cells were subsequently treated with 20, 40, 80, 160, 300 and 400 µM ferruginol for 24 and 48 h at 37°C, with vehicle control cells being treated with dimethyl sulfoxide instead of ferruginol. MTT solution (10 µl) was added to each well at 37°C for 2 h. The medium was completely removed and 500 µl dimethyl sulfoxide was added to solubilize MTT formazan crystals. The optical density was determined at 570 nm using an ELISA plate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

OVCAR-3 human ovary cancer cells were seeded at a density of 2×10⁶ cells per well and exposed to various treatment doses (0, 20, 80 and 300 µM) of ferruginol and incubated for 48 h at 37°C. Cells treated only with pure dimethyl sulfoxide served as vehicle control. Following trypsinization and washing with PBS, cells were stained with acridine orange/ethidium bromide (AO/EB) double stain (1 µl of each 5 mg/ml AO and 3 mg/ml EB stock solution). The cells were then washed with PBS, fixed in formaldehyde (10%) and the again washed with PBS prior to analysis using a fluorescence microscope at ×400 magnification (Nikon Corporation, Tokyo, Japan).

OVCAR-3 human ovary cancer cells were seeded in a flask at the density of 2×10⁶ cells per well and subsequently treated with various doses (0, 20, 80 and 300 µM) of ferruginol for 48 h at 37°C. Cells were then harvested and washed with PBS twice prior to the addition of 2.5% glutaraldehyde and fixation for 3 h at 37°C. The cells were embedded in an LR White resin for 30 min (Sigma-Aldrich;Merck KGaA) at 37°C. Following embedding, the resin block was sectioned using an ultramicrotome (JEOL, Ltd., Tokyo, Japan) and sections of 50–70 nm thickness were collected. TEM was performed using a transmission electron microscope (JEOL, Ltd.) at ×400 magnification. Apoptosis evaluation was performed by examining the ultrastructural cell changes in ferruginol-treated cells.

The wound healing assay was performed as described previously (9). Briefly, OVCAR-3 cells at a density of 2×10⁵ cells/ml were seeded in a 6-well plate and incubated at 37°C to attain a 100% monolayer of confluent cells. After starving cells for 24 h, a 50 ml pipette tip was used to create a straight cell-free wound in the wells. Subsequent to washing with PBS three times, the cells were treated with varying doses (0, 20, 80 and 300 µM) of ferruginol for 48 h at 37°C. Cells were subsequently fixed and stained with 3.5% ethanol containing 1.5% crystal violet dye for 20 min. Using an inverted light microscope (Nikon Corporation), 10 randomly selected fields were photographed and the fraction of cells that migrated into the scratched area was determined visually.

OVCAR-3 human ovary cancer cells were seeded at 2×10⁵ cells per well in 60-mm plates and treated with 0, 20, 80 and 300 µM ferruginol for 48 h at 37°C. Subsequent to drug treatment, cells were subjected to trypsinization and washed twice with PBS. Cells were fixed with 70% cold ethanol overnight and treated with 20 µg/ml RNase A at 37°C, which was followed by staining with 10 µg/ml PI at 37°C. The DNA content and cell cycle distribution was analyzed by flow cytometry using a FACSCalibur instrument (BD Biosciences, San Jose, CA, USA).

Data are presented as the mean ± standard error of the mean of at least three independent experiments. The differences between groups were analyzed by Student t test and one-way analysis of variance (in case of comparisons between >2 groups) using GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, CA, USA). *P<0.05 and **P<0.01 were considered to indicate a statistically significant difference.

Ferruginol belongs to the meroterpene class of natural products and has a phenolic structure (Fig. 1). The cytotoxic effects of ferruginol against OVCAR-3 human ovary cancer cells were evaluated by an MTT cell viability assay at various doses and incubation durations. The results presented in Fig. 2 indicate that ferruginol inhibited the growth rate of OVACR-3 cells in a dose- and time-dependent manner. Cells that were exposed to higher doses and higher treatment durations exhibited lower cell viability (Fig. 2). The IC₅₀ (half maximal inhibitory concentration) provides an indication regarding the effectiveness of a substance in inhibiting a specific biological or biochemical function, and IC₅₀ values for 24 and 48 h treatment durations were determined to be 175.2 and 84.6 µM, respectively.

In this assay, fluorescence microscopy using AO/EB staining was employed to investigate apoptotic effects induced by ferruginol in OVCAR-3 human ovary cancer cells. Results presented in Fig. 3 demonstrated that untreated control cells did not exhibit any red/yellow fluorescence, which indicated no signs of apoptosis. However, cells that were treated with 20, 80 and 300 µM ferruginol began to exhibit red/yellow fluorescence, which indicated the onset of the apoptotic process. Furthermore, it was observed that the amount of these apoptotic cells increased with increasing concentrations of ferruginol.

TEM was also used to investigate the apoptotic effects of ferruginol in OVCAR-3 cells. The results demonstrate that untreated control cells exhibited intact cellular nuclei with undamaged nucleolus (Fig. 4A). However, cells that were treated with increasing concentrations (20, 80 and 300 µM) of ferruginol exhibited chromatin condensation and disappearance of the nuclear envelope. At higher doses of ferruginol, apoptotic body formation was observed (Fig. 4B-D). Therefore, TEM results indicate that ferruginol induced apoptosis-associated morphological changes in OVCAR-3 human ovary cancer cells.

An in vitro wound healing assay was performed to investigate the effects ferruginol on cancer cell migration. The results indicated that the untreated cells did not exhibit any inhibition of cell migration after 24 h incubation with ferruginol. However, after 24 h treatment with 80 and 300 µM ferruginol, a dose-dependent inhibition of OVCAR-3 cancer cell migration was observed (Fig. 5). The percentage of migrated cells decreased from 98.7% in control to 68.2 and 45.3% in 80 and 300 µM ferruginol-treated cells, respectively. The number of cells that migrated into the scratched area were photographed before and after drug treatment, at 0 and 24 h.

The effects of ferruginol on cell cycle phase distribution were observed by flow cytometry using PI as a probe. The results presented in Fig. 6 demonstrated that the number of cells in the G2/M phase of the cell cycle increased in a dose-dependent manner between 0 and 300 µM. The percentage of cells in the sub-G1 phase also increased in a dose-dependent manner. However, the percentage of cells in the G0/G1 phase was decreased in ferruginol-treated groups compared with the control group.