HeLa, SiHa, C-33A and CaSki human cervical cancer cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The HeLa, SiHa and C-33A cells were cultured in Eagle's minimal essential medium (EMEM) and the CaSki cells were cultured in Dulbecco's modified Eagle's medium (DMEM), all of which were supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), penicillin (100 U/ml, Gibco; Thermo Fisher Scientific, Inc.) and streptomycin (100 µg/ml, Gibco; Thermo Fisher Scientific, Inc.), and maintained in a humidified incubator with 5% CO₂ at 37°C.

For radiation treatment, cells were grown and treated with or without osthole (see below for details) and then subjected to 6 Gy (the comet assay) or 10 Gy (western blot analysis) X-ray irradiation at a dose rate of 3.38 Gy/min using X-320ix (Precision X-Ray, Inc., North Branford, CO, USA) at room temperature.

The cells were seeded into 96-well plates at a density of 1×10⁴/well and grown for 24 h and then treated with different concentrations of osthole (0, 40, 80, 120, 160 or 200 µM; Chengdu Must Bio-Technology Co., Ltd., Sichuan, China) for 24 or 48 h at 37°C. At the end of each experiment, 5 mg/ml MTT in phosphate-buffered saline (PBS) was added and the cells were cultured at 37°C for 4 h. The cell culture supernatant was removed and 150 µl dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals for 10 min, following which the optical density was measured at 490 nm using a spectrophotometer (PerkinElmer, Inc., Waltham, MA, USA). The experiments were performed in triplicate and repeated at least three times. Data are summarized as the percentage of the control.

The cells were seeded into 6-well plates at a density of 1,000/well, grown overnight and then treated with different concentrations of osthole (0, 50, 100 or 200 µM) for 12 days. The culture medium was refreshed every other day. At the end of the experiments, the cells were stained with 1% crystal violet solution for 20 min at room temperature. Cell colonies with ≥50 cells were counted using an inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany). The experiments were performed in triplicate and repeated at least three times. Data are summarized as the percentage of the control.

The apoptotic rate of cells was measured using the fluorescence-activated cell sorter (FACS) following staining with the Annexin-V FITC kit (BD Pharmingen™; BD Biosciences, San Diego, CA, USA). The cells were grown in 6-well plates and treated with or without osthole for 24 h, and then collected for staining with the FITC-labeled Annexin V and PI kit according to the manufacturer's protocol. The cells were subsequently analyzed using the FACS Accuri C6 flow cytometer (Genetimes Technology Inc., Shanghai, China). The experiments were performed in triplicate and repeated twice. Data are summarized as the percentage of the control.

The cells were seeded onto chamber slides (Corning Inc., Corning, NY, USA) and treated with 100 µM of osthole for 24 h. Following treatment, the cells were washed with ice-cold PBS to remove detached cells and then fixed in 95% ethanol for 15 min. Following brief drying, the chamber slides were stained with 5 µl AO/EB (50 µg/ml), according to the manufacturer's protocol, and cell images were captured using a Leica DM 14000B microscope with digital camera (Leica Microsystems GmbH). The experiments were performed in triplicate and repeated twice. Data are summarized as the percentage of the control.

The cells were grown to reach 90–95% confluency in 6-well plates. The cell monolayer was wounded using a sterile 100-µl pipette tip and then washed with cell growth medium to remove the detached cells. The cells were cultured in serum-free medium and treated with osthole at different concentrations (0, 20 or 40 µM) for 24 h. Images of the wounded monolayer were captured at different time points using an inverted microscope (Olympus Corp., Tokyo, Japan). The experiments were performed in triplicate and repeated three times. Data are summarized as a percentage of the control.

The cells were grown and treated with osthole (0, 20 or 40 µM) for 24 h and then suspended in cell solution, and 2×10⁴ cells in serum-free EMEM were added to the upper insert of the Transwell chamber (Corning Inc.). The insert membrane was pre-coated with or without 50 µl Matrigel Matrix (1 mg/ml; Corning Inc.). EMEM supplemented with 20% FBS was added to the bottom of the Transwell plates, and the Transwell plates were incubated at 37°C for 24 h. The tumor cells remaining on the upper side of the membrane were removed using a cotton swab, and tumor cells that invaded the reverse side of the membrane were fixed with 95% ethanol and stained with 1% crystal violet solution for 20 min. Images were captured in five random microscopic fields at ×200 magnifications using an inverted Olympus microscope (Olympus Corp.). The experiments were performed in triplicate and repeated twice. Data are summarized as a percentage of the control.

The cells were seeded onto coverslips, treated with osthole (0 or 40 µM) for 24 h and then fixed in 4% paraformaldehyde for 10 min at room temperature. The cells were then permeabilized in 0.05% Triton X-100 in PBS for 10 min at room temperature and subsequently incubated with a monoclonal rabbit anti-E-cadherin (cat. no. 3195) or vimentin (cat. no. 5741) antibody at a dilution of 1:100, or a monoclonal rabbit anti-NF-κB p65 (cat. no. 8242) at a dilution of 1:200 at 4°C overnight, all from Cell Signaling Technology, Inc. (Beverly, MA, USA). The following day, the cells were washed with PBS three times and then incubated with a secondary anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor^® 555 conjugated; cat. no. 4413; Vector Laboratories, Inc., Burlingame, CA, USA) at a dilution of 1:500) at the room temperature for 2 h, and the cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc.). Cell images were then captured in five random microscopic fields using an Olympus fluorescence microscope (Olympus Corp.) (magnification, ×200). The experiments were performed in triplicate and repeated three times. Data are summarized as a percentage of the control.

Following the indicated treatment, the cervical cancer cells were collected, resuspended, and then loaded onto agarose coated glass slides, which were coated with 150 µl of 0.5% agarose, at a density of 1.5×10³ cells/µl. The slides were then soaked in a lysis buffer (10 mM Tris-HCl, 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100 and 10% DMSO) for 1 h and washed with neutralization buffer for 5 min, each for three times. The slides were then placed into an iced-cold electrophoresis solution (300 mM NaOH and 1 mM EDTA) and subjected to 25 V at 300 mA electrophoresis for 25 min. At the end of the experiments, the cells were stained with an ethidium bromide solution (20 µg/ml), and images were captured using an Olympus fluorescence microscope (Olympus Corp.). The numbers of cells with or without comet tails were counted and averaged. The experiments were performed in triplicate and repeated three times. Data are summarized as a percentage of the control.

Total cellular protein was extracted using a protein extraction kit (Thermo Fisher Scientific, Inc.), and protein concentration was measured using the BCA protein kit (Thermo Fisher Scientific, Inc.). The denatured protein samples of 30 µg each were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and electrophoretically transferred onto polyvinylidene fluoride membranes (BioTrace; Life Sciences, Port Washington, NY, USA). For western blot analysis, the membranes were blocked in 5% skimmed milk solution for 1 h and then incubated with a specific primary antibody at 4°C overnight. The following day, the membranes were washed with PBS-Tween-20 three times and then incubated with an anti-rabbit or mouse secondary antibody at the room temperature for 2 h. The primary antibodies were rabbit monoclonal antibodies against Bcl-2 (cat. no. 3498), Bax (cat. no. 14796), cleaved caspase-3 (cat. no. 9664), cleaved caspase-9 (cat. no. 20750), vimentin (cat. no. 5741), N-cadherin (cat. no. 13116), E-cadherin (cat. no. 3195), β-catenin (cat. no. 8480), MMP-2 (cat. no. 40994), MMP-9 (cat. no. 13667), Phospho-ATM (Ser1981; cat. no. 13050), ATM (cat. no. 2873), Phospho-Histone H2A.X (Ser139; cat. no. 2577), Histone H2A.X (cat. no. 7631), NF-κB p65 (cat. no. 8242), Phospho-IKKα (Ser176)/IKKβ (Ser177) (cat. no. 2078), IKKα (cat. no. 2682), Phospho-NF-κB p65 (Ser536; cat. no. 3033), NF-κB p65 (cat. no. 8242) and NF-κB1 p105/p50 (cat. no. 12540; all from Cell Signaling Technology) and used at a dilution of 1:1,000, while the secondary antibody was an anti-rabbit IgG SA00001-2 (Proteintech, Wuhan, China) and used at a dilution of 1:5,000. The protein bands were subsequently visualized using chemiluminescence (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and x-ray films.

Data are summarized as the mean ± standard deviation (SD) and were statistically analyzed using GraphPad Prism software version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA). Analysis of variance and Student's t-test were used to compare the values of the test and control samples. P<0.05 was considered to indicate a statistically significant difference.

To assess the anti-cervical cancer activity of osthole, a cell viability MTT assay was performed, and it was found that osthole reduced the viability of the four cervical cancer cell lines in a dose-dependent manner (Fig. 1A). In addition, the tumor cell colony formation assay showed that osthole treatment dose-dependently inhibited growth of cervical cancer cells (Fig. 1B). Morphologically, osthole treatment also reduced HeLa and SiHa cell-to-cell contact, and the osthole-treated tumor cells had more filopodia and cell layers (Fig. 1C).

The present study assessed whether the reduction in tumor cell viability was due to the induction of apoptosis using Annexin-V and FACS analyses. Osthole treatment significantly increased the apoptotic rate of the cells, compared with that of cells in the control group (Fig. 2A). To confirm the osthole-induced tumor cell apoptosis, HeLa cells were stained with AO/EB. In the control group, the uniform green staining showed normal morphology of cells, whereas osthole treatment induced early apoptosis of the tumor cells, which had condensed chromatin and red apoptotic bodies (Fig. 2B). At the gene level, osthole treatment increased the B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 ratio, and the levels of cleaved caspase-3 and cleaved caspase-9 in a dose-dependent manner, indicating that osthole activated the caspase-dependent pathway (Fig. 2C).

A previous study revealed that osthole suppressed the invasion capacity of lung cancer cells (13). In the present study, the effect of osthole on regulating cervical cancer cell migration and invasion was further examined using Transwell and scratch assays. The data showed that wound healing was significantly suppressed following osthole treatment in a dose-dependent manner (Fig. 3A). The Transwell assay data further supported this finding, as osthole suppressed the tumor cell migration and invasion capacity of the HeLa and SiHa cells (Fig. 3B).

Treatment of the cervical cancer cells with 50 mM osthole increased the expression of E-cadherin but decreased the expression of Vimentin in the HeLa cells, as evidenced by the immunofluorescence analysis (Fig. 4A). This indicated that osthole suppressed cervical cancer cell EMT. The expression of other EMT biomarkers, including N-cadherin, β-catenin, MMP-2 and MMP-9, were also analyzed (Fig. 4B and C), and the results suggested that osthole suppressed cervical cancer cell EMT. The osthole-induced suppression of cell adhesion proteins MMP-2 and MMP-9 in the HeLa and SiHa cells (Fig. 4C) also further support the results of the invasion and migration assays.

Radiotherapy is an important treatment option for locally advanced cervical cancer. A primary mechanism of irradiation is to induce tumor cell DNA damage (15). In the present study, a Comet assay was performed to assess the effects of osthole in combination with radiation on cervical cancer cells. The pretreatment of HeLa and SiHa cells with osthole (50 µM) for 24 h and exposure to 6 Gy radiation markedly increased the irradiation-induced cervical cancer cell DNA damage, compared with that in the control cells (Fig. 5A). At the protein level, this treatment combination significantly inhibited phosphorylation of the ataxia telangiectasia mutated (ATM) and γH2AX proteins, whereas no significant changes in the levels of ATM and H2AX were observed (Fig. 5B). These results indicated that osthole inhibited the irradiation-induced DNA damage repair capacity of cervical cancer cells.

Previous studies have shown that NF-κB signaling is involved in the regulation of tumor cell DNA damage (15) and that the osthole-induced suppression of lung cancer cell invasion capacity is mediated by suppression of the NF-κB-induced expression of MMP-9 (13). Therefore, it was hypothesized that osthole-enhanced cervical cancer cell DNA damage and inhibited tumor cell migration and invasion are also be mediated by manipulating the NF-κB signaling pathway. The HeLa cells were treated with osthole (0, 50, 100 or 150 µM) for 24 h and western blot analysis was performed to detect key proteins of the NF-κB signaling pathway. It was found that osthole treatment significantly reduced the phosphorylation of inhibitor of NF-κB (IκB) kinase (IKK)α and p65 proteins in the cytoplasm, but did not alter the levels of total IKKα or p65 (Fig. 6A). In addition, osthole treatment decreased nuclear protein expression of p50 and p65 (Fig. 6B). The subcellular localization of p65 protein in HeLa cells treated with osthole (0, 50, 100 or 150 µM) was assessed by immunostaining and light microscopy, and the data were consistent with the results of the western blot analysis (Fig. 6C). These results suggested that osthole inhibited irradiation-induced DNA damage repair in HeLa cells through suppressing NF-κB signaling.