Contributed equally
Osthole (7-methoxy-8-isopentenoxycoumarin) is an O-methylated coumarin, originally extracted from Chinese herbal medicine. It has been demonstrated that osthole has antitumor effects in various cancer cells
Human cervical cancer presents a significant worldwide health burden, particularly in developing countries (
Osthole (7-methoxy-8-isopentenoxycoumarin) is a monomer compound that is extracted from
In the present study, the antitumor activity of osthole in cervical cancer was investigated
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 (
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 (
A previous study revealed that osthole suppressed the invasion capacity of lung cancer cells (
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 (
Radiotherapy is an important treatment option for locally advanced cervical cancer. A primary mechanism of irradiation is to induce tumor cell DNA damage (
Previous studies have shown that NF-κB signaling is involved in the regulation of tumor cell DNA damage (
In the present study, the antitumor activity of osthole in cervical cancer cells was assessed
Osthole has been reported to induce apoptosis and have anti-proliferative activity in a variety of human cancer cells, including ovarian (
Tumor cell EMT is an important event during normal cell transformation into malignant cells and cancer metastasis (
Locally advanced stages of cervical cancer are usually treated with chemotherapy and/or a combination of external beam radiation therapy and brachytherapy (
Finally, the present study also demonstrated that osthole attenuated the activation of IKKα and IκBα, and decreased the translocation of p50/p65 into the cell nucleus, which further supports data from a previous study showing that osthole inhibited the NF-κB-mediated expression of MMP-9, lung cancer cell migration and invasion (
In conclusion, the present study initially assessed the anti-cervical cancer activity of osthole
Not applicable.
The present study was supported in part by a grant from the National Natural Science Foundation of China (no. 81473452).
All supporting data and materials are available within the article.
YC and JL performed experiments; ZL and JL and SW analyzed data; YY, LZ and KZ conceived and designed the experiments; YC wrote the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
phosphate-buffered saline
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
epithelial-mesenchymal transition
dimethyl sulfoxide
human papillomavirus
fluorescence-activated cell sorter
4′,6-diamidino-2-phenylindole
Osthole inhibits cervical cancer cell viability and proliferation. (A) MTT assay. HeLa, SiHa, C-33A, and CaSki human cervical cancer cells were treated with or without osthole (0, 40, 80, 120, 160, 200 or 240 µM) for 24 or 48 h and then subjected to the cell viability MTT assay. Osthole suppressed cervical cancer cell viability in a dose-dependent manner. (B) Colony formation assay. Cells were grown and treated with 50 µM osthole for up to 12 days and images were captured. Tumor cells with ≥50 cells were counted and the data revealed that osthole inhibited colony formation of HeLa and SiHa cells in a dose-dependent manner. *P<0.05 compared to the control cells. (C) Morphology. Tumor cells were grown and treated with or without osthole (0, 40, 80, 120, 160, 200 or 240 µM) for 24 h and, and images were captured using an inverted microscope with an attached digital camera at ×200 magnification. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Osthole induces cervical cancer cell apoptosis. (A) HeLa and SiHa cells were grown and treated with osthole (0, 50, 100 or 150 µM) for 24 h and subjected to the apoptosis assay. (B) Tumor cell AO/EB fluorescence staining. HeLa cells were grown and treated with osthole (0, 50, 100 or 150 µM) for 24 h and subjected to staining. (C) Western blot analysis. Tumor cells were grown and treated with or without osthole (0, 40, 80, 120, 160, 200 or 240 µM) for 24 h and then subjected to western blot analysis of Bcl-2, Bax, and cleaved caspase-3 and −9 proteins. *P<0.05 compared to the control group. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Cl, cleaved.
Osthole inhibits cervical cancer cell migration and invasion. (A) Tumor cell scratch assay. HeLa and SiHa cells were grown to ~95–100% confluency, scraped with pipette tips, and treated with osthole (0, 20 and 40 µM) for 24 h. Wound healing was observed and images were captured. Images show that osthole inhibited HeLa and SiHa cell migration in a dose-dependent manner. (B) Transwell tumor cell migration and invasion assays. HeLa and SiHa cells were grown and treated with osthole (0, 20 and 40 µM) for 24 h and then subjected to Transwell migration and invasion assays. Images show that osthole inhibited HeLa and SiHa cell migration and invasion compared with the control group (magnification, ×200). *P<0.05 compared to the control cells.
Osthole suppresses cervical cancer cell EMT. (A) Immunofluorescence analysis. HeLa cells were grown and treated with or without 50 µM osthole for 24 h and then subjected to immunofluorescence analysis of EMT biomarkers (magnification, ×100). The data showed that the expression of E-cadherin was increased, whereas that of vimentin was decreased in HeLa cells. (B) Western blot analysis. Tumor cells were grown and treated with or without osthole (0, 20, 40 and 80 µM) for 24 h and then subjected to western blot analysis for detection of EMT biomarkers vimentin, N-cadherin, E-cadherin and β-catenin proteins. (C) Western blot analysis. HeLa and SiHa cells were grown and treated with or without osthole (20 and 40 µM) for 24 h and then subjected to western blot analysis detection of MMP-2 and MMP-9. Expression levels of MMP-2 and MMP-9 decreased in a dose-dependent manner with osthole exposure (*P<0.05, vs. control). EMT, epithelial-mesenchymal transition; DAPI, 4′,6-diamidino-2-phenylindole; MMP, matrix metalloproteinase.
Osthole induces cervical cancer cell DNA damage induced by radiation. (A) Comet assay. HeLa and SiHa cells were grown and treated with 50 µM osthole for 24 h and then subjected to 6 Gy irradiation and then subjected to the Comet assay. A total of 50 cells were randomly quantified from the images and the percentage of cell tail length was calculated. Data are summarized as the mean ± standard deviation. *P<0.05 compared to the control. (B) Western blot analysis. HeLa cells were grown and treated with osthole (50,100 or 150 µM) for 24 h and exposed to 10 Gy radiation and then subjected to western blot analysis for the detection of p-ATM, ATM, γH2AX and H2AX. ATM, ataxia telangiectasia mutated; p-, phosphorylated; IR, irradiation.
Osthole promotes NF-κB signaling in radiation-induced cervical cancer cell DNA damage. HeLa cells were grown and treated with osthole (50, 100 or 150 µM) for 24 h and exposed to 10 Gy radiation. Cytoplasmic and nuclear proteins of HeLa cells were separately extracted for western blot analysis of key proteins in the NF-κB signaling pathway, including (A) IKKα, p-IKKα, p65, p-p65 and (B) p50. (C) Subcellular localization of p65 in HeLa cells was examined by analysis with a fluorescence microscope (magnification, ×50). All results are expressed as the mean ± standard deviation (*P<0.05, vs. control). NF-κB, nuclear factor-κB; IKKα, inhibitor of NF-κB kinase α; p-, phosphorylated; IR, irradiation; DAPI, 4′,6-diamidino-2-phenylindole.
Osthole sensitization of radiation in inhibition of cervical cancer cell viability by targeting multiple signaling pathways. Cervical cancer cells were grown and treated with osthole in combination with radiation. Combination treatment suppressed the protein expression of vimentin, N-cadherin, MMP-2 and MMP-9, induced the cleavage of pro-apoptotic caspase, inhibited the phosphorylation of γH2AX, IKKα and p65 proteins by ATM, and promoted the translocation of NF-κB from cell nuclei to cytoplasm. IR, irradiation; NF-κB, nuclear factor-κB; IKKα, inhibitor of NF-κB kinase α; p-, phosphorylated; ATM, ataxia telangiectasia mutated; MMP, matrix metalloproteinase.